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· THE EFFECis· OF MATERNAL ISOLATION ON THE ONTOGENY.OF

CIRCADIAN"ACTIVITY RHYTHMS AND THE GROWTH OF RAT PUPS

By

.VEANNE N. ANDERSON, B.Sc.

'A Thesis

SUQrnitted to the School of Graduate Studies

in Partial Fulfi~ment of the Requirements

for the Degree-~. -

Doctor of Philosophy

McMaster University

© April, 1985

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MATERNAL ISOLATION AND THE CIRCADIAN RHY'IJlMS OF RAT PUPS,•

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DOCTOR OF PHILOSOPHY (1985)(PsycholoF)

McMASTER UNIVE~SITY

Ham1lton~ Ontario

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TITLE: The Effects of Maternal Isolation on the Ontogeny ofCircadian Activity Rhythms and the Growth of Rat Pups

AUTHOR: Veanne N. An4er~on, B.Sc. (Colorado State University)

SUPERVISOR~ Professor G.K. Smith

NUMBER OF PAGES: x, ISS,

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Abstract

The circadian rhythms of young animals can be entrained by

prenatal and postnatal maternal cues. The present research

examined the effects of early postnatal matetnal isolation ~n the-

ontogeny o~ the act~vity rhythm in rat pup~. The studies also

examined the effects of different light cycles, as well as other

stimuli which may serve as synchronizers of the activity rhythm

during the postnatal period. Activ~ty rhythms synchronized toa

light-dark (LD) cycle.appeared as early aa five days after birth in

mother-reared pups. This result contrasts with data from other

reports whic~ indicate a late~ onset. Pups reared without their

mothers (AR pups) between ,3 or 4 to 18 days. postnatally on a LD

cycle had rhythms which were of lower ~plitude and of shorter

duration than those in the mother-reared group. AR pups with a LD

cycle and a feeding cycle which approximated the normal nursing

rhythm sh~~~ more synchronization of their activity than pups with

only a LD or feeding cycle. The mean period of the activity

rhythms of pups-raised under c~nstant light deviated the most from

24 hrs. The introduction of a temperature cycle attenuated the AR

pups' activity. These results indicate that the nursing rhythm, in

conjunction with LD cycles, may serve as synchroni~ers of rat pups'

rhythms. However, nursing and LD cycles represent only a part of

the complex postnatal environment which includes temperature, as

well'as other stimuli not investigated here, such as olfacto~y

cues.

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There was also evidence suggesting that rhythmic factors

during the early postnatal period may influence gr~wth. AR pups

with a cyclic feeding schedule had, ~eavier spleens and lighter

livers than animals with a noncyclic schedule. Heavier forebrains

were associated with a predominantly diurnal fe~ding cycle. These

growth factors may, in turn, influence the rhythmicity of locomotor~

activity.

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Acknowledgements

Research and the preparation of ,a dissertation are rarely

conducted in isolation from the cooperation and collaboration of

other pe?ple. The present experiments and manuscript are not

exceptions. First and foremost, I want to thank my advisor, Dr.

Grant Smith, for his generosity and support throughout all stages

of my graduate career.

Thanks also go to Dr. Harvey Weingarten 'for his comments and

to all of the people in the workshop, particularly E. Mitchell who

was responsible for building and helping with much of the equipment

used in the present experiments. In addition, Peter Northcott

taught me the artificial rearing technique and designed some of the

equipment. I would also like to thank Dr. Greg Brown for his ,suggestiona and the use of his laboratory. Associated with his lab

are Mahendra Joshi and Tim Burns who helped with many stages ~f the

pineal NAS study reported in Appendix F. In addition, Barbara

Graham provided aasistance in the design and execution of the'I

pineal NAS study. I am also grateful to Bev Bardy for help with

some of the revisions.

Most importantly, this dissertation is dedicated to Eric, my,

best friend, scientific colleague, and partner in life. He endured

the hardships and shared th~ pleasures of my doctoral work.

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I rTable of Contents

Abstract .' .

Acknowledgements ...........•.................. ' .

List of Figures .

List of Tables 0" ••••••••••• : •••

Chapter 1 - General IntroductionBackground ................•......................Ontogeny of Biological Rhythms •••• '.:•.•••••••••.Maternal Synchronization and Entrainment ••.••••••Outline of Experiments " .

Chapter 2 - Growth of Artifically Reared RatsI~ troduction .Methods •••••••••••••••••••.••••••••••••••••.•••••Resul ts .

. Discussion••••••••••••••••••••••••••••.••••••••• ,

Chapter 3 - The Development of Circadian Act~vity RhythmsIntroduction•••••••••••••••••••• ;' ••••••••••••••••Methods .Result8 •••••••••••••••••••••••• · ~ ••.•.••••Discussion ~ .

Chapter 4 - General DiscussionRhythm Development ••.•.••.•.•..•••.•••..•....••.••Rhythm Development and Grow~h••••••••••••••••••••

'-;" ' Implications for Human Infants ••••• '.' ••••••••••••. '-" ,~ummary••••••••••••••••••••••••••••••••••••••••••

,-Reference Note's ••••••••••••••••••••••••••••••••••••••••••••••

References ' .

Appendix A - Feeding Schedules r ' ..Appendix B - Autocorrelation analysis ••••••••••••••••••••••••

'.Appendix C - Aurocorrelation data ••••••••••••••••••••••••••••

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1624

273039 •44

57636883

100106108109

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136

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~able of Contenta (cont'd)

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Appendix D - Plota of 3-hr meana ••••••••••••••••••• , ••••••••• 145

,Appendix E - Open-field behavior ••••••••.••• '. • • • • • ..... • • • • • • • • • 147

Appendix F - Pineal NAS study of. 153

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Figure

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List of Figures

Follows Page

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A rat pup with the gastric cannula"in'place.I

Mean body weights of the AR and MRLD pupsand the control littermates.

Mean body Weig~S ~t weaning.

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Mean relative humidity in the water bathincubators."" 64

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Drawing of the activity transducer. 64

The 24-hr mean activity for the various groups. 69

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16-20

21-25

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27-31

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The proportion of dark-period activity forthe various,groups.

The proportion of AR animals food deprivedduring each 3-day block.

The proportion of ~ups in the various groupsshowing significant adtocorrelation peaks.

The mean periods of the activity rhythmsfor the various groups.

Frequency distributions of the,periods ofthe activity rhythma for the AR pupa.

Autocorrelation peak areas for the variousgroups.

Time of occurrence of peak 3-hr meanactivity for the vcriouB groups.

Frequency distribution of the time of,occurrence of 3-hr mean peaks of activity.

Three-hr means of activity for the variousgroups.

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Figure

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List of Figures (cont'd)

Ekample of an autocorrelation plot.

•Daily 3-hr mean activity for individualpups ,form the various groups.

Follows Page

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146

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Average daily body weights for mother-reared,intermittently-fed AR, and continuously-fed"AR pups. I

Average time spent g~ooming for control.IAR'and CAR pups.

Average head raises for control, tAR. andCAR pups.

, .Average ,lines crossed for control, !AR, and.CAR pups.

Average pivots for control, IAR, and CARpups.

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, List of Tables

Tablej

1 Artificial diet.

2 Sample sizes, humidity, incubatortemperatures and survival rates.

3 Mean organ weights.

~ Mean organ weights for the independentvariables in the regression analysis.

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5 Stepwise regression results for organweight data.

,--- 6 Average age at eye opening.

7 Experimental condLtions and water bathtemperatures_

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Sample sizes for the activity data.~

Mu\tiple R2s and slopes for'the ~inea~regression on 24-hr activity.

Stepwise regression results for the,proportion of dark-period activity. ~

!Deviations of the'proportion of dark-periodactivity from 50%.

Occurrence or autocorrelation peaks.

Mean periods for non-FD and FD autocorrelationpeak!! ..

Partial correlations between the organ weightsand the rhythmicity scores.

Mean age at appearance of developmentalindices

N-acetylserotonin (NAS) levels in pinealglands.

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Chapter 1

General Introduction

Background

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\TerminoloJ\Y\

Bi~l rhythms ,are ubiquit~us in both the animal and plant

kingdoms and affect such diverse prOcesses as pupal eclosion, predatory

hunting, and le~rning and memory. Biological rhythms may be defined as

biological processes which recur or vary in intensity at predictable

time intervals (Rusak & Zucker, 1975). The length of the recurring

time interval, referred to as the period (AscHoff. 1979), is used to

classify rhythms 'into three general types. Infradian rhythms have

periods greater than approximately 28 hrs and' encompass phe~omena sucl>'

as female reproductive cycles and se3sonal breeding in some mammals.

Ultradian rhythms can be represented by sleep-state rhythms and feeding

bouts (Bowden, Kripke, & Wyborney, 1978; Daan & Aschoff, 1981) and have

~peri~s less than about 20 hrs. Rhythms with periods of approximately

24 hrs (Halberg & Lee, 1974), Or circadian rhythms,'will be the major

focus of this dissertation. Rhythms are also described by other

pa~ameters. The peak of a rhythm refers to its maximum value whereas

the trough refers to the rhythm's minimum value. A rhythm's amplitude

is the dlfference between the peak value and the tro~gh value .•

Circadian rhythms can be ~ntrained by rhythmic stimuli, c~lled

zeitgebers Or ".time giving". stimuli (Borbeley, 197B~. When entrained,

a circadian rhythm maintains a stable phase relationship with the

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zeitgeber (Enright, 1981b). For example, when entrained to a zeitgeber

such as the light-dark (LD) cycle, a rat's peak of activity might

consistently occur 1 hr after the onset of darkness. The 1-hr lag

between the onset of darkness and the activity peak would be the phase

relationship. When rhythmic data are fit to a cosine function, the

term, acrophase, is used to describe the phase relationship between the

zeitgeber and the function's peak (Enright, [981b). Several zeitgebers

have been identified; for example, environmental pressure cycles

(Hayden & Lindberg, 1968), social cues (Takahashi & Murakami, 1982),

feeding schedules (Bolles & Stokes, 1965; Krieger, 1979; Spiteri,

1982), and most notably, 1ight-dark cycles One factor determining the

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entrairting effectiveness of a zeitgeber is the circadian rhythm itself.

Body temperature rhythms in monkeys are more readily entrained by LD

cycles whereas urinary potassium excretion rhythms are more stable 'with

a feeding cycle (Moore-Ede & Sulzman, 1981).'

Besides entrainment another property of circadian rhythms is•

their ability to freerun, or maintain their rhythmicity, in the absence

of cyclic input from zeitgebers (Aschoff, 1981). Locomotor activity

rhythms in rats (Richter,1971) and cortisol rhythms in humans (Milea,

Raynal, & Wilson, 1977) freerun under conditions of constant darkness

(DD). Rhythmicity is also maintained under conditions of constant

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light (LL), altho~gh under prd{Onge~ conditions, circadian rhythms

may "disappea,," and ultradian rhytnms may predominate (Albers, Gerall,

& Axelson, 1981; Houma & Hiroshige, 1978). The periods of freerunning

circadian rhythma deviate slightly from 24 hrs (entrained conditions)

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and may depend on the lighting conditions present before placement on

LL or DD (Davis & Menaker, 1981).

Analysis of Rhythmic Data

A variety of methods are available for the a~alyses of rhythmic

data (Enright, 1981a). The two types of analyses used for the rhythm

data presented in Chapter 3 will be described here. Day-night

differences in a set of data are frequently used to establish

rhythmicity. This measure is particularly usefu~ for determining

entrainment of a rhythm to a LD cycle when, the rhythm has defini~e

peaks or troughs during the light or dark period. Nonsign11icant

day-night differences do not necessarily imply a lack of rhythmicity or

even entrainment. In these cases the rhythm may be freerunning or

entrainment may be obscured by the manner in which the data were

sampled and/or combined for' analysis.

Time series analyses 'are more sophisticated ways of determining. .

if rhythmicity is present in a set of data. In general, a prerequisite

for the use of time series analyses is a long series of data consisting

of many "daily obse~ations (Enright, 1981a). One of the more simple

analyses is autocorrelation analysis. This involves the calculation

of a product-moment correlation "between the original data series, and

that seme series when it is 'lagged' on itself by some fixed number of

time units" (Enright, 1981a, p.27). Usually a large number of lags are

'examined and the correlations are then plotted against the lag. If a

stable rhythm is present in the data, the plot of the autocorrelations

against the lags will be, cyclic and the lags of the peaks will

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correspond to the dominant period of the data (Enright, 1981a).

Problems in interpreting the results of autocorrelation analyses will

be discussed in Chapter, 3. Unlike some time series analyses,

autocorrelation analysis does not assume a priori the shape of the'

function which best 'describes the data.

Physiological Aspects

Physiological studies of circadian rhythms have focussed on the

'visual system (Rusak & Zucker, 1979). Transections of the, optic nerves

and ,bilateral enucleatlon do not disrupt free running circadian

rhythmicity in mammals, although entrainment to a LD cycle is lost

(Davis & Menaker, 1980; Deguchi, 1975b; Moore, 1975; Richter, 1971).

Lesions of the primary optic tract and/or the accessory optic tract do

not impede LD entrainment in rats (Moore, 1975) although reentrainment

to a shifted 10 cycle may be retarded in hamsters (Rusak ~ Boulos,•

1981). Similar results are seen when the lateral geniculate nuclei, a

'major terminal site for visual fibers, are 1esioned (Dark & Asdourian,

1975; Rusak & Boulos, i981).

The suprachiasmatic nuclei (SCN) , tWf hypothalamic nuclei

located dorsally to the optic

rhythmicity. ~~ SCN receive

,chiasm, are important to circadian

direct input from the retina via the

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retinohypothalamic tract (RRT) (Picka~Silverman, 1980; Sawaki,

1977). Lesions of the SCN in rats and hamsters disrupt a variety of

ci~cadian rhythms (Redgate, 1976; Saleh & Winget, 1977; Stephan &

Nunez, 1977) although animals may continue to show ultradian rhythms

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