Effect of prenatal heat stress on brain growth and serial discrimination reversal learning in the guinea pig

Brain Research

Bulletin, Vol. 1, pp. 133-150,

All rights of reproduction

in

1976.

Copyright

0 ANKHO

InternationaI

Inc.

any form reserved. Printed in the U.S.A.

MONOGRAPH Effect of Prenatal Heat Stress on Brain Growth and Serial Discrimination Reversal Learning in the Guinea Pig’*“?’ KEITH M. JONSON, JACK G. LYLE, Department of Psychology MARSHALL J. EDWARDS AND RICHARD H. C. PENNY4 Department of Venterinary Medicine University of Sydney, Australia (Received

13 August 1975)

JONSON, K. M., J. G. LYLE, M. J. EDWARDS AND R. H. C. PENNY. Effect af~r~nata~ heat stress on brain growth and pig. BRAIN RES. BULL. I(1) 133-150, 1976. - Exposure of pregnant guinea pigs to an environmental temperature of 42°C for 1 hr daily on Days 20-24 of gestation resulted in a significant reduction in the birthweight and brainweight of newborn offspring. These deficits persisted to early maturity and were still evident at 250 days of age following behavioral testing. Although some degree of postnatal neurogenesis and brain growth had occurred, this was not sufficient to compensate for the retarding influence of prenatal hyperthermia. Heat stress was also observed to seriously impair learning performance on the original discrimination task and this tendency persisted over the subsequent 4 reversals for both initial and perseverative errors. Impaired learning performance was related to reduction in brainweight. Animals with lower brainweights made significantly more initial, perseverative and total number of errors over the 5 problems. In addition, 12 of the 14 behaviorally tested stressed progeny had brainweights that were at least 2 standard deviations below the mean of the controls and as a result were classified as micrencephalic. Although heat stressed animals showed a significant reduction in the number of amacrine synapses in the inner plexiform layer of the retina, performance differences were not attributed to changes in synaptic organisation of the retinal circuitry or to visual or other ophthalmic defects, but brain function. Animals with higher mean maternal poststressing core temperatures gave birth to progeny which had smaller whole and part wet-br~nweights. Examination of the effect of poststressing core temperature on brainwei~t revealed that brain growth, independent of bodyweight, was retarded when this temperature elevated above 41S”C. This represented a rise of approximately 2.1”C above normal and for each 1°C rise above this temperature, brainwei~t was reduced by 0.4227 g of the control value. Analysis of the atmospheric content of the incubator during stressing sessions ruled out the possibility that changes in level of oxygen and carbon dioxide may have contributed to the retarding influence on fetal development and learning ability in the guinea pig. serial dis~~i~~nat~on reversal leam~~~~n theguinea

Heat stress Prenatal hyperthermia Critical period Vulnerability of developing embryo Teratogenic effects Postnatal brain development Retina Micrencephaly Learning performance impairment Behavioral testing

Guinea pig brain growth rates Early gestation Brainweight deficit Brain growth Synaptic organisation Serial discrimination reversal learning

EXPERIMENTAL hyperthermia induced during certain critical periods of fetal development has resulted in a significant increase in incidence of congenital malformations in newborn guinea pigs [ 14, 16, 171, rabbits 181, rats [ 15,461, mice 136,421, sheep [301 and primates 1431. The adverse effects of maternal hyperthermia on the prenatal development of the brain has been extensively investigated in the guinea pig [ 14, 17, 18, 20, 211. In these studies the procedure for inducing maternal hyperthermia .____ ’ Requests for reprints should be sent to Keith M. Jonson, Clinical

was to place pregnant animafs in a forced-draught electric egg-incubator set at 42-43’C for 1 hr daily on 1, 2,4 or 8 consecutive occasions. In general, this resulted in a mean maternal-core temperature which peaked at approximately 3.6 C above normal. The nature and severity of the teratogenic effects were dependant upon the stage of gestation at which heat stress was applied, the number of exposures and degree of maternal hyperthermia induced. In the guinea pig, the incidence of developmental abnormal-

Psychology Unit, Department of Psychology, University of Sydney Sydney, N.S.W. 2006, Australia. ‘Thanks are due to Mr. P. Beaumont, M. D., Division of Ophthalmology, University of New South Wales, for conducting the ouhthalmoloeical examination. 3This research was in part supported by grants from the Australian Research Grants Council and Smith Kline and French Laboratories. 4Present address: Department of Veterinary Medicine, The Royal Veterinary College, University of London, Hatfield, Herts, England. 133

133

JONSON. LYLt., f l>WAKDS AND t’k:iiNY

ities was highest following hyperthermia on Days 18 to 75 and included exomphalos, hypoplasia of digits and incisors. muscle and skeletal defects. renal agenesis and reduced bodyweight. At certain stages of gestation. maternal hyperthernlia was also associated with moderate to severe wet-brainweight deficits in newborn offspring [ 14, 16, 17 j Mcun brainweight adjusted for differences in bodyweight for newborn guinea pigs heat stressed on 8 consecutive occasions between Days 4 to 67 of gestation are sumnlarlsed ~11 Fig. 1. Progeny with an adjusted brai~weight 2 SD below that of the control group were diagnosed as micrencepl~allc 1171. The results indicate that brainwei~ht was most severely retarded and the incidence of micrencephaly higtiest following exposure to hyperthermia on Days 18 to 25. Fine-grain experimental analysis of this critical period revealed that prenatal brain development was most vulnerable to retardation following heat stress on Days 30 to 21 or 22 to 2.1 of gestation [ I6,17]. It was subsequently shown that reductlon of whole brainweight by hyperthermla on Days 20 to 23 of gestation, was accompanied by proportional deficits in the wet weights of cerebral hemispheres and of the brainstem and cerebellum. Furthermore, the DNA content in each brain segment was significantly less than for control values indicating a reduction in cell numbers, possibly neurons [ 2 1 I . In these studies, animals were sacrificed within 12 hr ot birth. Thus it was not known to what degree heat stress during the most critical stage of brain development might affect learning performance in the mature offspring, or Lo what extent postnatal neurogenesis would compensate prenatal retardation of brain growth evident at birth. A small degree of postnatal neurogenesis is known to occur in the guinea pig [ 21. The aim of the present investigation was to examine the effects of exposure to heat stress on Days 20 to 24 of gestation on brain growth and serial (iiscrimination reversal (SDR) learning performance in guinea pig offspring at maturity. The effects of heat stress during the critical stages of mid and late gestation were also examined and reported elsewhere 13’71. METHOD

Preparation

of Heat Stressed aad Controi Anlnrals

Experrmentai (Cauta poreelks)

females.

Forty-five female guinea pigs of heterogeneous stock were used. Animals weighed between 520 and 930 g. Food and water was available ad lib and diet was supplemented daily with greens to meet their daily Vitamin C requirements 1351. The colony was housed in a temperature controled environment of 21 (*2)“C with a relative humidity of 50 to 70 percent. Animals were equated for bodyweight and 32 were allocated to the experimental group and 13 to the control group. These were combined with a group of guinea pigs from a related project 1371, and randomly alocated to 8 pens. Bodyweight was recorded weekly. Mating. From 16 days prior to the commencement of mating, females were examined daily for estrus. The onset of estrus was determined by complete breakdown of vaginal closure membrane [ 171. Seven days prior to the commencement of mating, a fertile male was placed in an open-wire-mesh cage in the pen in which it was to be used. ~~~g~a~~y diagnosis. Following the introduction of the

FIG. 1. Mean adJusted brainweights tar rtewborn guinea l)igsheaL on 8 consecutive occasions between Days 4 I,., h? .?f gestation (* mean based on 4 hra~~wei~htsonly ).

shwxxi

male, pregnancy was considered ttj ILWC commenced the first day on which complete breakdown of the \agmal membrane was recorded (Day 0 of pregnancy). Animals were diagnosed as pregnant following a failure of recurrent of vagmal membrane breakdown wlthin 18 days of the 1ns1 recording of the estrus onset. The mean length of the-estruk cycle for the gumea pig 1s 16.3 I +O.YY1 days. Pregnancy WI> subsequently confirmed by palpation of tense spherlcdl fetal vesicles within the abdomen During gestation, females were exammed daily tl)l evidence of abortion or recurrence of estras. On Day 60 of the 68 day gestation penoa, pregnant females were mdlvldually caged and checked twice daily until psirturition Determination of body temperalure. Ikep body temperature was recorded immediately before and. after exposure to experimental hyperthermia with a .ciinical thermometer. A lubricated thermometer was placed at least 8 cm into the animal’s rectum and this position wa9 maintamed ttntll no further temperature increments occ~urred. Procedure for inducing h~~er~flert~l~. Maternal hypcrthermia was experimentally induced by placing tiimals in a forc~d”draught electric egg-incubator itnimals were caged in perforated polythene boxes 41:~ 28 x 15 cm and were exposed daily for I hr ofi Days 30 tri 74 of gestatioir. The incubator was set at 42.0°C dry bulb and regulated between 22.0 to 29.0°C wet bulb. The internal air space of t-he incubator was 0.77 m’. Air circuiation in and out 31’ the incubator was aided by fans which drew in air via 4 ports each 4.5 cm in dia. Dry and wet bulb readings were taken at 15 min intervals. Treatment of control animnis. (‘ontrol animals wc’ri! treated m a manner similar to heat-stressed animals except that for 1 hr daily on Days 20 to 24 they were placed in an incubator which was maintained at. room temperature. E{ousing of newborn of~~pnng. &lit stressed an-d contra! offspring were raised in the same-pen. Guinea pig young-are reared communaliy and this permitted opportunity for fostering and cross-fostering control- 1353. Hay. guinea P@ pellets and water were available ad lib and fresh green lucerne was supplied twice per day of’oxygen and Carbon Dioxide During Heating

&&rm&ation Incubator

()n each of 4 randomly

Ikvels

zri the

selected sessions, the ~i~In~s~h~~e

PRENATAL

HEAT

STRESS

ON BRAIN

GROWTH

AND LEARNING

of the experimental incubator was sampled after 0, 15, 30, 45, and 60 min. The control incubator was similarly sampled following 0 and 60 min of exposure. Samples were collected in tonometer flasks and the contents were analysed by the Polargraphic Oxygen Electrode Technique and the Infra-red Carbon dioxide Technique.

Determination

of the Effect

of Hyperthermia

135

auditorv disturbances were minimized in view of previous findings. [35] Apparatus. Guinea pigs were tested on a specially designed discrimination box [ 341 The essential features of the apparatus are illustrated in Fig. 2.

on Develop-

men t to Birth Within 12 to 15 hr of birth, progeny were weighed and examined for external abnormalities. Twenty-one animals were killed by exsanguination with pentobarbital sodium anesthesia. The brain, including the olfactory lobes but not the pituitary, was dissected from the skull, sectioned from the cord at the atlantoepistrophic articulation and weighed immediately to the nearest milligram. of the Effect of Prenatal Hyperthermia Brain Development at Early Maturity and Maturity

Determination

on

Eighteen animals were sampled at early maturity while 28 others were sampled after behavioral testing. The animals were killed and their brains removed and weighed as described previously. In addition, the cerebral hemispheres were separated by section in the midline through the corpus callosum and dissected from the brainstem by vertical section through the stria terminalis. The cerebellum was dissected from the brainstem by section of the cerebellar peduncles. Brain segments were placed in tared tubes and weighed separately to the nearest milligram. Effects of Prenatal Hyperthermia

on the Eye and Fundus

Ophthalmic defects in guinea pigs exposed to hyperthermia during early gestation have previously been reported [ 14,161 These included microphthalmia, cateracts, strabismus, blindness, and detached retina coloboma, (Edwards and Mulley, unpublished observeration). Defects of this nature impair photoreception and would influence visual discrimination learning performance. An ophthalmological examination was carried out to determine the incidence of defects in heat stressed and control animals immediately prior to behavioral testing. Animals were premeditated with homatropine (2 percent) and neo-synephrine (10 percent) 1 hr prior to the examination. The fundus of each eye was systematically examined by indirect and direct ophthalmoscopy for retinal abnormalities and optic nerve atrophy. The clinical examination reveaieci no ophthaimic abnormalities in any of the heat stressed or control animals Determination of the Effects of Prenatal Hyperthermia on visual Serial Discrimination Reversal (SDR) Learning in the Mature Offspring Animals. Seven male and 7 female guinea pigs were randomly selected from each of the heat stressed and control groups for the study. The remaining animals were employed in another, as yet unpublished behavioral study, [32]. Animals were approximately 130 days old at the beginning of the study. Mean bodyweight was 450 g for heat stressed and 501 g for control animals. They were housed, fed and watered in individual cages in an airconditioned laboratory of 18 (+2)“C. Lighting was maintained on a 12 hr day-night cycle. Sources of extraneous

FIG. 2. The Apparatus: A, starting compartment; B vertical sliding guillotine door; C, choice area; D, and D, ,brass rods for operation of Plexiglas swing doors E, and E, respectively; F, and F,, discrimination alleyways; G, clear Plexiglas dividing partition; H, metal grid; I, and I,, one-way swing doors (discriminanda); J, and J, , doorways; K, food compartment; L, food dish. The overall dimensions of the discrimination box were 86 x 40 cm. The starting compartment was separated from the discrimination chamber by an opaque guillotine door. This provided access to 2 discrimination alleyways divided by a clear Plexiglas partition. The alleyways could be individually closed off by clear Plexiglas doors hinged near the start-box entrance. A pair of swing doors were located at the end of each alleyway. These served as the discriminanda and required the animals to at least make contact with the stimulus to be discriminated so that access could be gained to the food compartment. Each pair of doors were mounted on a movable frame so that the lateral position of the positive and negative discriminanda could be reversed. Experimental procedures. Details of experimental procedures and the rationale for their adoption have been described elsewhere [ 34,35 1. Adaptation to food-deprivation schedule. Animals were initially provided with a 4-week laboratory habituation phase. Over the next 6 weeks, animals were gradually adapted to a 21 hr food-deprivation schedule. Drinking water was available ad lib. During adaptation, the daily supply of fresh vegatables was provided in a liquid form 1 hr prior to feeding on guinea pig pellets. Synthetic ascorbic acid was incorporated in the liquid vegetable diet to meet their daily Vitamin C requirements. Animals were handled daily for approximately 5 to 10 min during feeding. Habituation and pretraining. Adaptation to the experimental apparatus was facilitated by permitting access to all compartments over 7 30-min sessions. During these and all subsequent sessions, liquid food (cabbage) reinforcement was only available in the food cup of the apparatus. Once goal box training was established, animals were given up to 10 massed reinforced trials per day. The 2 sets of mid-grey colored doors were successively closed a little more on

136

JONSON, LYLE, ~I~WAR~S

AND PENNY

each trial so that on the seventh day ot training, ail animals Determinatron vf the Ejyect of PFrizutai Hyprrthermk2 oii had learned to nose open at least 1 set of closed doors to Retmal Development gain access to the food cup. The effect of prenatal hyperthzrmra on the hrstology Preliminary training. This training phase provided animals and dry weight of the retina was assessed foltowing with experience in opening both sets of doors. Five pairs of behavioral testing. trials were provided on each of IS successive days. Each Preparation of retinue jar light ctmi electron mtcroscwpy. pair of trials was comprised of a free trial followed by a Immediately prior to removal ot‘ the brain from ancstheforced trial. In a forced triaf, the entrance to the discriminatized animals, the right eye of ~ch animal was enttoieated. tion alleyway previously chosen was closed off thereby hemisected anterior to the ora serrata and the lens removed directing the animal to the alternative alleyway. At the end Surplus vitreous was scooped out and the posterior hemrof each days session, animals were permitted to feed freely sphere immersed in 3 percent o;smtum tetroxide. buffered at the food cup for approximately 90 sec. In this and all with 0.1M phosphate buffer (ptl 7.4 t 0 07) lor 1 !>Isubsequent sessions, the ascorbic acid content of the During the first 5 min of fixation. the retina was gently reinforcer was adjusted so that animals received at least teased from the pigment epitheliurn. Retinae were progrestheir daily Vitamin C requirement. sively dehydtated in a graded ethanol-water series, emVisual SDR problem training. Problem training was accordbedded in aralditr and allowed to hanlcn at 60°C‘ ing to a modified noncorrection procedure. The mid-grey Thick sectrons ( 1 -2 ,urn) were i:irt from 5 segments of colored doors were replaced with a set of black doors and :t each retina, stained with toluidmtl blue and photographed set of white doors. Black was adopted as the posrtive together with a graticule on a %ersx I’l~otomi~ros~~pe The stimulus for all animals on the original disc~minat~on retinal thickness between the ~)titet limitmg anil rnnc‘r (Reversal 0). The lateral position of the positive and the limiting membranes was measured with vernier cahpers on negative discriminanda was changed from tnal to trial enlarged micrographs (X ?5OI. Iritrathm sections apptoxiaccording to the Gellerman [ 291 orders. mately SO- X0 nm in thickness, wtre collected on ZOO-mesh The animals were given 10 massed trials per day and naked grids, stained with uranyl acetate and lead citrate and each trial terminated with a reinforced response. A trial examined with Hitachi HI11 1I? ;rnd Philips ?Ol I:lectron consisted of placing the animal in the starting compartment microscopes. and raising the guillotine door 3 set later. If the animal Dcterminatwn of’dry retimzi wtght. Following removaL nosed open the doors of the positive discrrminandum, 5 set of of the right eye. the left eye ~zcls similarly cnucleated, feeding was permitted. If the negative discrrminandum was hemisected and the lens removt%d. Surplus vitreous was approached, the opposite swing door was closed locking the removed from the posterior hemisphere and the retina animal in the incorrect alleyway and the response was carefully teased away from the pigment eI~Ithel~um The recorded as an initial error. An animal was permitted up to retina was immersed in a specumen ruhe ~ontain~~~~absolute 3 incorrect (perseverative) errors on any one trial. Foilowethanol and placed in a dehydratii)n oven at 60 C’. Wh?n ing the third incorrect response, the animal was forced to weight decrements were no longer evident, tubes were make a correct response by closing off the incorrect placed m a chromic acid bath Tubes were then dehydrated. discrimination alleyway. Training continued 7 days a week reweighed and dry retinal weights I:sl~ulated with each animal progressing at its own pace. Reversal training commenced when an animal had KESULTb attained a criterion of 9 out of 10 errorless trials on each of ?. successive days. Reversal training consisted of a series of problems each constituting a reversal of the previous Mean oxygen and carbon droxtdz percentages sun~&d problem. On Reversal 1, for example, the set of white doors over the 4 one-hour sessions appear m Table I. became the positive dis~riminandum and so on. Training At commencement of exposure_ the oxygen level W;I~ contunued until all animals had learned to criterion a total normal. It showed a slight but msignificant decrease m the of 5 problems. ‘I’ABLL 1 MEAN

OXYGEN

AND

CARBON

DIOXIDE

SAMPLED

OVER

PERCENTAGES E‘OIJR ONE HOUR

OF

INCUBATOR

ATMCISPHE,RI;

SESSIONS

Normal

experimental Oxygen Carbon dioxide Control oxygen

atmosphere

0 mm

20.93

20.93 (iOr 0.07 (?0.01731

0.03

20.93

Carbon

dioxide *Readings not taken.

0.03

20.93 (LOI 0.03 (to)

mill

30 nun

45 nnn

00 min

20.82 ~CO.04691 0.07 (to.02541

20.80 (iO.01731 0.07 (iO.03)

20.79 iiO.02241 0.18 (*0.03321

?fi.7 3 I iO.02651 It.24 (+1X052)

I

*

*

‘0.93

.3

3.

*

0.07 (~0.0141)

is

PRENATAL

HEAT

STRESS

ON BRAIN

TABLE

GROWTH

2

THE EFFECT OF HYPERTHERMIA

ON GESTATION

Heat Stressed (exposed to 42°C)

Control (exposed to 21°C)

Number of females mated

32

13

Number of females pregnant

32

13

Treatment

Stage of gestation on which treatment was applied Number of treatment sessions Duration of treatment

TABLE 5

5

1 hr

1 hr

EFFECT

Mean maternal poststressing core temperature

42.99”C (tO.2221)

39.56”C (tO.7051)

Mean elevation during treatment

3.59”C

0.08”C

to treatment

2

0

Number of accidental maternal deaths

7

1

Number aborting

9

0

Number of females delivering litters at parturition

14

12

Number of offspring delivered at parturition

46

39

8

3

38

36

heated incubator over the exposure periods. The initial level of carbon dioxide in the experimental incubator was higher than normal and was probably a by-product of heat reacting with urine and excreta from previous heatings. A slight rise in carbon dioxide level was evident after the first half-hour of exposure. Control carbon dioxide levels also rose slightly during the hour’s exposure. Such slight increases in atmospheric carbon dioxide levels are probably of little significance as guinea pigs expire between 4 to 6 percent carbon dioxide [ 481. It was concluded that the slight decrease in atmospheric oxygen and increase in carbon dioxide would not contribute to any extent to the effects that might be observed in the experimental offspring. on Gestation Days

20 to

24

PRENATAL

Abnormalities noted Hypoplasia of incisors Hypolasia of digits Talipes Exomphalos

of females

The effects of treatment during gestation are summarized in Table 2.

OF

at Birth on The

of

3

HYPERTHERMIA OFFSPRING

Number of offspring examined Mean litter size Mean birthweight (g)

Number of maternal deaths due

Effect of Hyperthermia

on Development

Days 20-24

Days 20-24

39.47”C (kO.4911)

Number of offspring alive at birth

of Prenatal Hyperthermia

The effect of maternal exposure to hyperthermia general prenatal development was assessed at birth. results are presented in Table 3.

39.4O”C (iO.2280)

deaths

The 45 guinea pigs became pregnant by the second estrus cycle. Nine heat stressed and 1 control animal died during or following exposure and 9 aborted. Eight of these were due to accidental perforation of the rectum caused by a thermometer. Of the 46 offspring of heat stressed mothers, 7 were stillborn and 1 died shortly after birth. Three of the control offspring were stillborn. Effect

Mean maternal pre-stressing core temperature

Perinatal

137

AND LEARNING

ON

NEWBORN

Heat stressed

Control

46 3.06 91.09

39 3.00 100.7

5 4 1 1

0 0 0 0

Effect on brain development. Seven newborn animals were randomly selected from each of the experimental and control groups for this purpose. A bodyweight difference was found between the heat stressed (HS) and control (C) animals (Table 3) which suggested the need for a bodyweight control group (BWC). Each heat stressed animal was matched for bodyweight and sex with a newborn control animal. The brainweights and bodyweights of these groups are presented in Table 4. Wet weights of brain segments were subsequently examined and summarized in Fig. 10. These data were not analysed in view of previous findings [211. Examples of heat stressed and a control brain from animals of similar bodyweight is shown in Fig. 3. Hypotheses. In previous studies it was found that heat stress during early gestation (Days 18-25) resulted in a reduction of brain weight and bodyweight [ 17,211. Furthermore, brainweight of heat stressed offspring was significantly less than would have been predicted in terms of bodyweight. On the basis of these findings it was predicted that: (1) Bodyweight and brainweight of controls should be significantly greater than that of heat stressed animals and their bodyweight controls. (2) Significant differences in brainweight but not bodyweight would exist between heat stressed animals and their bodyweight controls. These hypotheses were tested by 2 separate sets of planned contrasts. The results of these analyses are presented in Table 5. The analyses indicate that the data support these hypotheses. Contrasts 1 and 3 show that both bodyweight and brainweight of controls was significantly greater than the average for bodyweight control and heat stressed groups. Contrast 4 shows that brainweight for bodyweight

.fONSON,

I38

TABLE 4 BODYWEIGHTS AND BRAINWEIGHTS OF HEAT STRESSED CONTROL AND BODYWEIGHT CONTROL GUINEA YIGS AT

Control

(N = 7)

(N = 7)

mean t SD. Bodywe~~t g Brainweight g

80.86 (+9.7101 2.314 (iO.2521

mean + SD 102.7 I (c18.OI 1 2.864

(iO.351,

I-nWARDS

AND

PENN\

-These groups were sampled lmmeillstrl~ belorz f~haviorJ testing of the age control and 11~31
Bodyweight control (N= 7)

lf_vpothesex Previous evidence i ,’ I 1 &monstrateLi iltdt whole brainweight deficit
mean I S.D.

though some postnatal neurogrnosis 15 known to o,cur In the guinea pig, it was expected that postnatal compen<;;itorg growth would not make up the &&it by maturity. On the

BIRTH

Heat stressed

LYlk.

80.7 1 (i3.781) 2.663 (io.nqI 1

controls

was significantly greater than for heat stressed even though differences in bodyweight were not evident (Contrast 2). animals,

The effect of prenatal hyperthermia on growth of the brain to early maturity was examined on a group of 6 contro1 and 6 heat stressed offspring which were matched in age and a group of 6 weight-control offspring which was matched to the heat stressed group in sex and bodyweight.

basis of this evidence it wab i:rcdi&d that: l Lk Fistmortem agr oi hodyweight uotltt!~? animals would ~!Jft‘er significantly l‘rom that oi controi ,~nklheat stressed anrmals, hut age differences would not t??Id between ~clnirol ~(1 heat stressed animals. (7) Body~tght whole ;+n(! part h~~inwelgh~s r)f control animals \\c>uld by sigrIIf‘icantly greater than that of heat stresst~i znd hodyweighi ur)ntrol animals. (3) Significant differer~;~~8 in whole :rnd par; brainweIghts, hut not bodyweigl~:. would zxict heiwz
‘The results obtamed tram UIX~\IS of thti dat;t Inchcate that heat stress during early pitation tesultcd in d significant reduction in bodywcrght :tt rarly maturity. Normal animals matched for hoJywzIght with hear stressed animals were approximately one-half the ,?gd cjf heat

FXG. 3. Brain of newborn guinea pig heat stressed on Days 20 to 24 of gestation on left -- brainweight I .69g. birthweight 78 g. Brain of newborn control on right - brainweight 2.57 g, birthweight 80 g.

PRENATALHEAT

STRESSONBRAINGROWTHAND

139

LEARNING TABLES

ANALYSES OF BODYWEIGHTS AND BRAINWEIGHTS (HS), CONTROL (C) AND BODYWEIGHT-CONTROL BIRTH

Source

Contrast

Bodyweight

1 2

2C-lBWC-1HS lBWC-1HS

3 4

2C-lBWC-IHS 1 BWC- 1 HS Error

tk0r

Brainweigh

t

*Significant tsignificant

FOR HEAT STRESSED (BWC) OFFSPRING AT

df

MS

F

1 1 18 1 1 18

2244.02 0.07 199.87 0.66 0.43 0.08

11.23t 1.0 8.681_ 5.46*

at the 0.05 level. at the 0.01 level.

TABLE6 AGE,

BODYWEIGHT AND BRAINWEIGHTS OF HEAT STRESSED, CONTROL AND BODYWEIGHT CONTROL GUINEA PIGS AT EARLY MATURITY

Heat stressed (N = 6)

Age (days)

Bodyweight

g

Mean + S.D. Control (N = 6)

Bodyweight Control (N = 6)

124.7 (i15.46)

127.2 (t5.34)

64.8 (i-15.46)

449.2 (k56.38)

630.5 (t44.65)

441.0 (?59.31)

Whole Brainweight g

3.313 (+0.4630)

4.334 (?O. 1019)

3.871 (+0.1873)

Left Hemisphere g

0.930 (t0.1166)

1.200 (r0.0519)

1.168 (10.0529)

Right Hemisphere g

0.944 (+0.1260)

1.252 (t0.0793)

1.161 (tO.0648)

Brainstem g

0.953 (?0.1410)

1.200 (fO.0734)

0.997 (tO.0678)

Cerebellum g

0.431 (iO.0830)

0.543 (?0.0400)

0.539 (~0.0458)

stressed animals. Whole brainweights, and weights of all brain segments except cerebellum from control animals, were significantly greater than the average weights for heat stressed and bodyweight control animals. Weights of wholebrain, hemispheres and cerebellum of the bodyweight control group were significantly greater than those of heated animals indicating that reduction in brainweight was not merely a product of general bodygrowth retardation. However, brainstem weights from heat stressed animals were not significantly different from those of their bodyweight controls. Ej@ct of Prenatal Hyperthermia on Visual Serial Discrimination Reversal (SDR) Learning in Mature Offspring Hypotheses. A priori, it was hypothesized that heat stressed animals would perform at an inferior rate to their controls by making more initial errors and perseverative errors over

the 5 problems. Sex differences were not expected on the basis of previous findings with this species [ 341. The results of 2 split-plot repeated measures analyses of variance carried out separately for initial errors and perseverative errors over the 5 reversals are shown in Table 8. Combined male and female initial errors to criterion for each of the 5 problems for heat stressed and control animals are presented in Fig. 4 and perseverative errors in Fig. 5. These analyses indicate that Between Group differences significantly favoured control animals in the mean number of initial and perseverative errors made in attaining prereversa1 criterion. The highly significant Between Problems effect revealed by each of these analyses demonstrated that substantial changes in learning performance occurred over the course of the 5 reversals. The characteristic nature of this effect is illustrated in Fig. 4. The significant Problem x Group interactions indicated that learning over the 5

I40

JONSON, TABLE

LYLE.

hDWARDS

AND PE.NNY

7

OF BODYWEIGHTS, BRAINWEIGHTS AND PART BRAINWEIGHTS I*OK HEAl (HS), CONTROL (C) AND BODYWEIGHT CONTROL (BWC) OFFSPRING Al f ARI,Y

ANALYSIS STRESSED

MATURITY .._-_I___

Control

Age

,Sourcc

I

Bodyweight

2HWC -I(‘ I(‘ -111s I~rr~~r

t

5

Right Hemisphere

Error(B) Within Animals Problems (P) PXG PXS PXGXS Error(w)

*Significant tsignificant

8030.99 855.15 137.93 211.40 248.39

at the 0.025 at the 0.001

11.11 x15:

9 10

2C-- 1BWC- 1 HS

1 1

16002670.0 14061680.(~ 897069.90

I7.84t 15.76”

lBW(‘L’rror

IHS

15

2<‘- lBW(‘~-IHS lBW(‘-IHS Error 2C-lBWC’-1HS IBWC -1HS Error

reversals

8

30.65t 1.00 1.00

111.34

level. level.

I9.ost 9 75*

1 I

I

I I 15 I I IS

0.20 571596.80 1006464.0 646 148.0 3498120 0 362702.1

X.16* I 1.78 9.64*

at the 0.01 level. at the 0.001 level.

Initial Errors MS l;

4 4 4 4 96

1.83 0.94 0 IIt

IS

916675.0 16983680.0 649559.30

IBWC--1HS E.rror

TASK

34039.21 941.20 67.21

is 97 i

1 I 15

13 14

1 1 1 24

1395 1.411 80.01( 3039.7h

I

2C- 1 BWCm- I HS

ANALYSIS OF INITIAL ERRORS AND PERSEVERATIVE ERRORS OVER 5 REVERSALS ON A VISUAL SDR LEARNING

Between Animals Groups (G) Sex (S) GXS

1

1

7 8

Cerebellum

df

xx,3xi-

Ii

11 12

TABLE

Source

14924.69 i x.7s i68.8h

l:rrs>r

Brainstem

*Significant tsignificant

I

MS

I I I .5

2(’ IHW(’ -IHS IBWC’ IHS

6

Left Hemisphere

111s

2C‘ I BWC- I I-IS 1 BWC’-- 1 HS l:rror

3 4

Whole brainweigh

dl

Perseverative MS

1097.60 35.00 17.86

Errors 1:

20.597 1.00 1.00

53.30

32.33t 3.44% 1 .oo 1.00

410.36 61.49 18.39 16.89 20.08

20.43t 3.06* 1.00 1.00

progressed at differential rates with making more initial and pcrseverative

heat

stressed

errors across the reversals. Sex differences were not evident in these analyses. Failure to find sex differences justified combining their results (Figs. 4 and 51. Posteriori analyses of the origmal discrimination problem revealed that heat stressed animals made significantly more initial errors (t = 2.89, il’f = 26, p
PRENATAL

HEAT

STRESS

ON BRAIN

GROWTH

loo-

learn phenomenon similar to that previously observed with this species [ 341. Nevertheless, at the end of the study, the heat stressed group was still performing at an inferior rate to the control group (Figs. 4 and 5).

90-

SO-

Effect

60-

0 &

50-

$

40-

z :

30-

!z

2c-

Heat

IO-

6

i

Stressed

0

Control

*

;

j

i PROBLEMS

FIG. 4.

Mean initial errors for heat stressed and control guinea pigs on a visual SDR learning task.

Heat Stressed

0

Control + 01

I

0

I

I

I

I

,

3

4

PROBLEblS PIG.

5.

of Prenatal Hyperthermia

on Retinal Development

Histological assessment of the retina

70-

iii

141

AND LEARNING

Mean perseverative errors for heat stressed and control guinea pigs on a visual SDR learning task.

effects was slower for heat stressed animals. The control group demonstrated negative transfer effects up to the first reversal, whilst the heat stressed group showed cumulative negative transfer of greater magnitude up to the second reversal. Thus heat stressed animals not only showed inferior learning performance on the original discrimination problem, but they also showed a greater degree of perseverative interference as a result of reversal of reinforcement contingencies. Following negative transfer, the cumulative positive transfer effect indicated that both heat stressed and control groups profited from experience and as a result made fewer initial and perseverative errors with each successive reversal. Progressive improvement across a series of such problems demonstrates evidence for the operation of the learning to

Light microscopy. Mean retinal widths were 105.83 (k12.24) pm for the heat stressed and 105.53 (k10.97) pm for the control animals. These differences were not significantly different (p>O.O5). Retinal abnormalities were not evident in the sections surveyed. Electron Microscopy. Electron microscopical examination of preparations from all heat stressed and control animals revealed that obvious abnormalities were absent and gross structural integrity of the retina appeared intact. Recent research has shown that certain environmental conditions are capable of modifying synaptic organisation in cortical neurons [ 10, 24, 25, 44, 49, 501. The possibility that prenatal hyperthermia might influence incidence of retinal synapses was investigated in a preliminary stereological study (Sosula and Jonson, unpublished data). Ten electron micrographs were taken at a constant magnification (X 25,000) of the inner plexiform layer of each retina. A representative micrograph of the inner plexiform layer of a heat stressed and control animal is presented in Figs. 6a and 6b respectively. Electron micrographs were enlarged to x 75,000, their order randomized and the number of amacrine and bipolar synapses counted by 2 independent investigators. The mean number of amacrine synapses in the inner plexiform layers of the heated group (0.0156/clm2) was approximately half the number in the control group (0.0297/pm2, p 0.05). Surplus vitreous adhering to retinae was not considered to have influenced these findings as percentage water in the vitreous is over 99 percent [ 111 and approximately 73 percent in the retina [7]. Thus prenatal hyperthermia does not appear to have affected dry retinal weight of mature offspring. Effect of Prenatal Hyperthermia Maturity

on Brain Development

at

At the termination of visual SDR problem training, animals were killed and the whole brain and brain parts were removed and weighed according to the procedure described previously. An example of a heat stressed and a control brain from guinea pigs of similar bodyweight is presented in Fig. 7. Mean values for age, bodyweight, brainweight and part brainweights at post-mortem examination for heat stressed and control animals are presented in Table 9. Percentage deficits of the mean control value for bodyweight, brainweight and part brainweights in heat stressed and control animals from birth to maturity is summarized in Table 10. Separate analyses of variance of age and bodyweight yielded significant differences between heat stressed and

FIG. 6. ial Efecfron ~ni~~~~~~~ sfm&g rqi:ginnof inner ykxexiforrnlayer from hit-stressed gkrirxe;rpig retina, ~rn~~~~~proees~ (AP, ) forms amacrine synapse (A,%. A~~acr~~e s~mq~es are charac&rked by a sickening in their pre- and postsynaptic membranes in assocWon with a dense ag~ega~jo~ UT synaptic vesicies (SV) apposirtg the presy~ap~ic memb~~e. The densely vesi&ated bipolar process (BP) forms a bipoiar,syn&pse (B). Bipolar synapses are characterized by a synaptic ribbon (SRI in their presynaptic cytoplasm. The bipolar dyad synapse cantains a synaptk ribbon surraundea by 3 halo oi vesicles, a p~sy~apti~ membrane ~ec~~~ation and two postsyna~f~c neural proeesses.(N~~ and Nk’, :&, The amacrine process (AP,) farms a reciprocal conventional synapse (A2 f onto the bipular +ocessS, Osmium Vexation: ~a~~~~cat~on 63,500. fb) Electron r~~~ograp~ &awing region uf inner plcxi~orm. layer from control guinea pig retina. The bipolar process (BP) forms two bipolar synapses 03, Hi f with prominent synaptic ribbons oriented toward their dyadic sites. The amacrine processes (AP, , .M$ ) form twa conventional synapses {A, +4, ) with dense aggregations of synaptic vesicles clustered close to their extensive_ presynaptic zones. Amacrine synapse A, is a w&-oriented synapse with a clearly d&fined rnemb~~e spec~~~~~~io~. Amacrine synapse R, has heen obfigueEy cut and its rno~br~~~ Vestie ckkster IS’.‘> Es Sot s~~3~~zat~or~ is rn~~~~y ~de~~~~ab~~. The membra~~ss~c~~r~~ su~c~en~i~ we% defined to be scored as a synapse. Note that ins~ider~e of. osrn~op~~~i~.&XO~WX granules (1;;) in both neural procisses and MuIfer neurngiiat processes (WP). ~~n~i~~~ Exatiion: ~a~~~~cat~~n 69,900.

PRENATAL

HEAT

STRESS

ON BRAIN

GROWTH

AND LEARNING

143

FIG. 7. Brain of mature guinea pig heat stressed on days 20 to 24 of gestation on left - brainweight 3.08 g, bodyweight 555 g, age 286 days. Brain of mature control on right - brainweight 4.30 g, bodyweight 560 g, age 220 days. control groups. The results of these analyses appear in Table 11. The difference in age between the 2 groups was due to control animals being killed off at a younger age. Although heat stressed animals were significantly older at time of post-mortem examination, they weighed substantially less than their controls. These differences suggested the need to adjust for their effects in subsequent analyses. Analyses of covariance with age at death and bodyweight as the covariates were carried out on brainweights and part brainweights for heat stressed and control groups. The results of these analyses are presented in Table 12. The analyses indicate that with these adjustments, significant differences still existed between heat stressed and control groups in whole and part brainweights. Sex effects were not significant. Incidence of Micrencephaly. The incidence of micrencephaly was determined according to a previously reported procedure [ 17 I A regression of brainweight on bodyweight was calculated for control animals: Brainweight g = 2.6988 + (0.0024.bodyweight g), R = 0.8138. The regression of brainweight on bodyweight with -2 standard deviations was constructed for the control population. Brainweights corresponding to bodyweights of heat stressed and control animals were plotted and are presented in Fig. 8. Animals whose brainweights were 2 standard deviations below those of controls were classified as micrencephalic. According to this procedure 12 of the 14 heat stressed and none of the control animals were considered micrencephalic. The inci-

dence of this condition was significantly different for the 2 groups: x2 = 17.19,p<0.001. Prediction of Brainweight. A stepwise multiple-regression equation predicting brainweight from poststressing core temperature, bodyweight and age at post-mortem for heat stressed and control groups combined is presented in Table 13. The results of this analysis indicate that a combination of temperature, bodyweight and age in that order accounted for approximately 81.4 percent of the variance. Of these, temperature was the best single predictor accounting for 67.4 percent. Sex accounted for an additional 0.5 percent of the variance and did not contribute significantly to the prediction. Relation between maternal temperature and brainweight. The relation between mean maternal poststressing core temperature and brainweight for heat stressed and control groups was assessed by means of second-order partial correlation coefficients in which the effects of age and bodyweight were partialed out. The results of these correlations are presented in Table 14. Temperature was found to be significantly related to whole brain and all part brainweights indicating that females with higher mean maternal poststressing core temperatures produced progeny with lower wet brainweights. Effect of maternal temperature on brainweight. The effect of maternal poststressing core temperature on brainweights of the heat stressed group was determined by multiple linear regression analysis: Brainweight g = 19.9055

TABLE AGE. AND TURK

9

BODYWEIGHT, BRAINWEIGHT, PART BRAINWEIGHI’S MEAN MATERNAL POSTSTRESSING CORE TEMPE:RAFOR HEAT STRESSED AND CONTROL GUINEA EMPLOYED IN THE BEHAVIORAL I-NVESTIGATION

PIGS

Heat stressed tN = 141 Ape (days)

246.0 (t25.9)

Bodyweight (g)

516.19 P.86. t )

(i IOO.5)

3 199 l+0.431f

4.09 l5Q.2241

0 933 (r&1425

0 ri (17X)

43.18 (+0.48h)

39.3X (CO.4S2)

Whole bralnweight (8)

592.9 *Significant nt the .05 level. ~Si~ni~cant at the 01 level. ~Si~nificant at the ,001 level

Left Hemisphere (g)

Right Hemisphere
Temperature (“(‘)

TABLE

I LS

10

PERCENTAGE DEFICITS OF MEAN CONTROL VALUE t-OR BODYWEIGHT, BRAINWEIGHT AND PART BRAINWEIGHTS 1N HEAT STRESSED AND CONTROL GUINEA t’IGS FROM BIRTH TO MATllRITY

Birth Age (days) Bodyweight (8) Whole brainweight (6) Cerebral hemispheres (g) Bninstem (g) Cerebellum (g)

0 21.27 19.19 20.60 19.62 22.94

t:urly maturity

Matunty

i 15

250

28.75 23S6 24.08 20.58 20.62

17.26 ‘1.78 27.6 1 19.12 30.43

+ l,665{bodyweight~ + (--.4IE7. temperature *C). K = 058. The results are diagrammed m Fig. 9. The data indicate that brainweight independently of bodyweight, was retarded when maternal poststressing core temperature exceeded approximately 41.S°C, an elevation of about ?.l*C above normal. For each 1°C elevation above this temperature, brainweight was reduced by 0.4227 g, which represented approximately 10.33 percent of the control value. Relatron Between Second-order

~~~~~wel~ht and Lrarnmg partial

correlation

coeificlents

Perjortnume controlling

FIG. 8. Brainweights corresponding ICIb~~~wel~hts for hpar stressed and control guinea pigs. Brainweights tiling 2 standard dev-iations below control regression line were clasnified as micrencephalic. the effects of age and hodywe~ght were calculated on bramweight with learning performance error scores. The results of these correfations appear in Table 14. With the exceptIon of brainstem and total perseverativti errors, alt hrainweights were significantly related to inittal and persevemtive error score totafs and the Sum of their ‘totals. This indicated that animals with lower wet-whole brain and e!art bramweights made more initial. persekrative and hence total errors on the 5 SDR problems. Predictton of feorning performarm?. A stepwrse;multiple regression equation predicting total learnmg~performance error scores from temperature, brsinwQht and zage al post-mortem for heat stressed and control:groups combined is presented in Table 15. The results of this analysis indicate that a combination of temperatu.r~., ~~ainwei~t and- age at post-mortem in that order accounted for approximately 68.1 percent of the variance. Of these, temperature again for

PRENATAL

HEAT STRESS ON BRAIN GROWTH AND LEARNING

145

TABLE 12 AND BODYWEIGHT FOR HEAT STRESSED AND CONTKOL

RESULTS OF ANALYSES OF COVARIANCE WITH AGE AT POST-MORTEM WEIGHT

AND

PART

BRAINWEIGHTS

Whole Source

df

Groups (G) Sex (S) GXS Error

1 1 1 22

“‘Significant isignificant SSignificant

Left Hemisphere F MS

brainweight I: MS

1.70 0.07 0.02 0.08

21.72$ 1.00 1.00

0.25 0.06 0.02 0.03

Right Hemisphere F MS

8.64 2.11 1 .oo

11.16-F 2.03 1 .oo

0.11 0.02 0.004 0.01

at the 0.05 level. at the 0.01 level. at the 0.001 level

AS THE COVARIATES GUINEA PIGS

Brainstem MS

0.07 0.00001 0.003 0.008

OF BRAIN-

Cerebellum 1: MS

1;

7.92* 0.00 1 1.oo

0.05 0.01 0.002 0.002

18.92$ 3.91

I .oo

proved to be the best single predictor and accounted for over 63 percent of the variance. A combination of temperature and brainweight accounted for approximately 65 percent of the variance. Addition of bodyweight and sex to the equation yielded R = .830 which added approximately 0.8 percent of the total variance. These 2 variables did not contribute significantly to the prediction of learning performance error scores. DISCUSSION

FIG. 9. The effect of maternal adjusted brainweights of mature brainweight is shown by arrow. g (IO.33 percent) for each 1°C

poststressing core temperature on guinea pig offspring. Mean control Brainweight was reduced by 0.4227 rise above approximately 41S”C.

The effect of experimentally-induced prenatal hyperthermia was examined on postnatal brain development and visual SDR learning performance in mature guinea pig offspring. Pregnant animals were exposed to an environmental temperature of 42°C for 1 hr daily on Days 20 to 24 of gestation. The effect of hyperthermia on postnatal brain growth in the progeny was examined at birth, early maturity and maturity. The results of the present investigation confirmed previous findings on the extreme vulnerability of the developing embryo to the teratogenic effects of heat stress. In general, the effects of hyperthermia on gestation, and the range and incidence of congenital abnormalities in the newborn offspring was similar to previous observations when it was applied between Days 18 and 25 of gestation [14, 17,211.

TABLE PREDICTION

OF BRAINWEIGHT

13

I:ROM TEMPERATURE,

STRESSED

AND CONTROL

BODYWEIGHT

GUINEA

AND AGE I;OR HEAT

PIGS

Coefficients Step number

Constant

Temperature

1

17.90

2

13.75

3

14.81

-.1341 (.0183)* F=53.61 -.I065 (.0162) F=43.05 -.1215 c.0196) I:=38.38

Brainweight

Age

+.0022 (.0006) F=15.62 +.0021 (.0006) F=14.69

*Figures in parentheses represent standard scores.

-.0025 C.0019) 1:=1.15

R

df

,821

1,26

53.61

.894

2.25

49.69

.902

3.24

34.70

I.‘

14h

PARTIAL CORRELATION CCJEPFICIEMTS UC BRAIN~~IGH’~ WXTH L~,
Total perseveratwc errors Total

initial and perseverative errors *Significant at the .OSlevel. tSignificant at the .01 level fSignificant at the .OOl level ‘FABLF. I5 PREDICTION OE LEARNING PERFORMANCE ERROR SCORES FROM TEMPLRA I tiRP, BRAINWEIGHT AND AGE FOR HEAT STRESSED AND CONTROL GUINEA PIGS

I

-2S96.08

2

-1690.28

3

--1058.70

+21.555s

(4.1323)” 1:=44.49 +X.7692 (7.1843) F=8.36 t13.6614 f 8.4088) I’=264

SO.6029 (43.9611 t 1:=1.33 -69.2341 (44.5438) 1~=2.42

+.83% (S‘t97, 1;=2.33

794

I,_76

34 49

g&l

? 7_._z

?3 1”)

.825

3,14

i !.J-!

“Figures in parentheses represent standard srures In previous studies, slight but nonsignificant deficits were reported in the birthweights of heat stressed offspring [ 17,2 I 1. However, in the present investigation prenatal hyperthermia was found to reduce both birthweight and brainweight by approximately 20 percent of the control value. Furthermore, the retarding influence of hyperthermia on brainweight was s~~if~cantly greater than would have been predicted in terms of birthweight. Although brains from heat stressed and control offspring were anatomically simifar m appearance, stressed brains were considerably smaller in size. Subsequent examination of wet weights of cerebral hemispheres, brainstem and cerebellum revealed that percentage weight deficits found in these brain segments were proportionately similar to those for the whole brain. These results suggest that all segments of the developing brain were approximately equally affected by the retarding influences of prenatal hype~hermia. The deficits in bodyweight and wet brainweight observed at birth were still evident m the heat stressed offspring at early maturity, immediately prior to behavioral testing. Separate examination of the hemispheres. brain-

stem and cerebellum revealed that ah brain segments from heat stressed animals weighed substantrally kss thc& theil controls. In general these were sign~~~ant~y Less than would have been predicted in terms of bodyweight. The ratio of percentage deficits in heat stressed -compared with control brainweights had increased slightly hut remained proportionately similar to that observed at b&h. Separate examination of the relative grow_th rates for whole and part brain weights summarized m Fig. 10 illustrate these findings graphically. These data clearly demonstrate that the degree of postnatal neurogenesis and. brain growth that had occurred, apparently was not suffmient to compensate for the retarding influence of prenatal hypertherrnia on postnatal brain development. From the results of the learning. experiment It 1s apparent that prenatal exposure- to hyperthermia during these critical days of gestation seriously impaired learning performance in the guinea pigs at maturity. Heat stressed animals demonstrated significantly inferior performance .on the origins1 discrimination task and this tendency persisted over subsequent reversals. This trend was evident-for bdth initial and perseverative errors made -in attaining prereversat

PRENATAL

147

HEAT STRESS ON BRAIN GROWTH AND LEARNING 5

WHOLE

BRAIN Control

l

Heat

0

4

CEREBRAL 0)

3 k (3

stressed

HEMISPHERES

Control

a

Heat

o

stressed

BRAINSTEM

2 2

Control

*

Heat

A

stressed

CEREBELLUM

1

I

0

r

125 AGE

Control

l

Heat

o

stressed

t

250

(days)

FIG. 10. Relative growth rates of wet whole and part brainwejghts surnrn~~~d for heat stressed and control guinea pigs from birth to maturity. criterion. Although heat stressed animals performed at a rate inferior to their controls, they nevertheless demonstrated evidence of learning to learn the simple visual SDR task. Unlike the brain, weight changes were not evident in the neurat retina. This suggests that this aspect of the nervous system is not susceptible to the same retarding effects of prenatal hyperthermia when applied on Days 20 to 24 of gestation. Although the gross retinal architecture appeared normal under light and electron microscopic examination, heat stress had interfered with the fine structure of the inner plexiform layer. A stereological analysis of the synaptic formation in this layer revealed a significant decrease in the incidence of amacrine but not bipolar synapses in mature and heat stressed animals. The functional significance of synaptic changes is not known. However, recent research has demonstrated visual performance on a simple light-detection task to be unimpaired in Wistar albino rats lacking photoreceptors and showing similar reductions in synaptic density of amacrine processes in the inner plexiform layer [33]. Although information processing at the level of the inner plexiform layer of heat stressed animals would be somewhat affected, this evidence suggests that it is untikely that a decrease in synaptic incidence influenced visual functioning to account for performance deficits observed on the SDR learning task. These performance deficits were apparently not attributable to other ophthalmic defects. In addition, the finding that heat stress has also been found to impair spatial SDR learning performance further suggests that the observed differences in learning ability were probably not due to visual deficits but brain function [ 321. The retarding effects of prenatal hyperthermia on developm~ut observed at birth and early maturity were still present at maturity following visual SDR problem training. Heat stressed progeny weighed less than their controls even

though they were approximately 35 days older. Brainweights and part brainweights adjusted for effects due to differences in age and bodyweight at post-mortem, also weighed significantly less for heat stressed than control animals. Twelve of the 14 heat stressed animals examined had bra~nweights that were at Ieast 2 standard deviations below the mean of the controls and as a result were classified as micrencephalic. Apart from obvious size differences, the gross anatomical structure of micrencephalit brains was not visibly different from that of control brains. In general, the ratio of percentage weight deficits of the control value for whole brain, cerebral hemispheres, brainstem and cerebellum was similar to that observed at birth and early maturity. The failure to observe further increments in relative growth rates from early maturity to maturity suggests that postnatal brain growth was complete for both heat stressed and control guinea pigs by Day 125 after birth. The cerebral hemispheres and cerebellum for example, showed little weight increase beyond Day 65 suggesting that, at least in normal guinea pigs, these segments mature earlier than brainstem. These results also demonstrate that retardation of brain development from prenatal exposure to hyperthermia was irreversible. A relationship was found between brainweights of stressed offspring and mean maternal poststressing core temperature. Females with higher poststressing core temperatures gave birth to progeny which had smaller whole and part wet-brainweights. Temperature also proved to be the best single predictor of brainweigbt at maturity and accounted for approximately 67 percent of the variance. Fine-grain analysis of the effect of maternal poststressing core temperature on brainweight at maturity revealed that braingrowth, independent of bodywei~ht, was retarded when this temperature elevated above 41.5”C. This represented a rise of approximately 2.l”C above normal. For

148

each l°C rise above this temperature, brainwe~ght was reduced by 0.4227 g. Evidence from the time course of core temperature elevation during stressing and poststressing recovery, suggests that temperature remained above this critical level for less than 30 min f 161. And, only m the final 15 min of the 60 min heating session did the maternal core temperature elevate above the critical point at which detectable reduction in brainweight was observed. Thrs evidence demonstrates that even within the group subJected to heat stress, the degree of maternal temperature attained above 41.5OC was a critical factor affecting brainweight of the progeny at maturity. A significant relationship was found to exist between impaired SDR learning performance and reduction in wet brainweight at maturity. The results indicated that animals with lower brainweights made significantly more initial, perseverative and total number of errors over the 5 problems. In general, this trend was also evident for cerebral hemispheres, brainstem and cerebellum. Temperature again proved to be the best smgle predictor of learning ability and accounted for approximately 65 percent of the variance. Thus exposure to hyperthermia during the critical Days 20 to 24 of gestation not only resulted m severe brainweight reduction but also seriously impaired SDR learning performance in the mature guinea pig offspring Analysis of the atmospheric content of the incubator during stressing sessions ruled out the possibility that changes in level of oxygen or carbon dioxide may have contributed to the retarding influence on fetal development and learning ability observed in previous studies with the gumea pig [4,6, 531. These performance differences couid not be attributed to factors other than the effects of hyperthermia induced during the stressing session. Recent surveys of the literature indicate that fetal development [3lj and subsequent behavior 131 can be affected by maternal psychological (emotional~ stress. The treated control procedure employed in the present investigation insured that both heated and control mothers experienced otherwise identical stressing treatments. A certain trend appears evident when the findings of the present study are compared with those of 2 other studies investigating the effects of prenatal hyperthermia on brain growth and reversal learning performance at maturity [ 371, For example, it was found that offspring exposed during the less critical Days 40 to 44 of ~ntra-uterine development showed no performance deficits on the original discrimination task, but a significantly greater number of perseverative errors on the first reversal. These performance deficits were subsequently found to be related to reduction of wet weight of brainstem and cerebellum [ 191. Cumea Pigs stressed on the least vulnerable Days of 56 to 60 of prenatat development, showed no performance deficits [37]. or detectable reduction in brainweight [ 191. Thus heat stress had its greatest influence in retarding brain growth and impairing learning and performance when exposure was instituted during the early critical stage of fetal development. These effects diminished with advancing gestation. The pathogenesis of brainweight reduction is not fully understood. The experimental evidence has shown that reduction in brainweight following exposure durmg this critical embryonic developmental period is associated with a decrease in DNA content, indicating a reduction in bra111 cells fzl] Although DNA cell estimate procedures do not diffe~ntiate between neurons and neuroglia, histological

JONSON, LYLE, EDWARDS AND l’E?&L anafysis showed that aeuronai pr(3~lterat~on was acti-vely taking place during this period ] 18 I This suggests that the cell deficits were largely neuronat t/~ficrt~. Studies on the ceI1 generation cycle of the centrai nervous systcril followmg one such exposure dunng tlus pt~r~oti. indrcatu that cell death and an inhibitory per&l ot furtl~er mrtotic ~~~~t~vrty could both contribute to the hr,i!n (~11 deficit> [ lOJ. Factors responstble for clfectmg t!l.:~ ;Ihnormalrtles h;r,se not yet been identified. A number of direct and mdrrc~..t ractors that may be important in providing retardation af’ bram growth have been suggested I I61 . For example, &ctructictn of fetal C~IIY in mitosis at the time of heating may be dut2 ti.) direct thermal msutt. App~cati(~n of iiircL~i heat to rat utcrinc horn while maternal temperature remamed unchanged, has reSulted in congenital malformatrons rncludrng hr:trn deformities 1461. similar to those reported when b,:al wa\ applied maternally [ 151 Indirect effects that may pIa> ;i con~ri~~ul.(~r~ toie include placental damage, metaboh disturbances caused 10 the mother, changes in electrofyti< concentrations. general maternal reactions to stress and loss &IImaternal weight Restriction of dietry intake durmg geststron doer not appear to have important ~~onst*~Irienccs, c)fl pt.lstrrataI offspring development or learning performance (0.311. Although a marked reduction in maternal apprtitc: had imviously been observed in female guinea pigs severely affected hy heat f 161 , exlrcmr tiic.rari_ testrtctlcm\ iiiiftrtg pregnancy were not associated \si? II congenitrtl ~thr)itrmalities in guinea pig offspnng f 13 1 Postnatal maternal undernourtslimttnt durmg kzcratrnn and i?~fantile nl~nutrition m ihe j nImi early prrrutl ot development have both been showrt to tntluenct bodyweight 139, 41, 471 and learning prrtormancc [S. 41. 371 An attempt was made to mimmi/.e the possibility ~,t undernutrition during weaning by permrtttng the opportunity for crossfostering in commun~ily housed stressed and control animals. Nevertheless, rt F. possible that stressed offspring were less successful m znmpeting with control offspring for maternal nutrition. Malnutrition w;is laoI a eontributmg factor as hay, guine,: pig pt‘llets ,md water were available ad lib and fresh green luccrne twits: pei day. However, the fact that percentage bodyweight dnd brainweight deficrts rernained prnportion~tely similar ~IUIN brrth to maturity. indicates that pus-tndtal undernutrition and lnalnutrition were not important influences in the pathogenesis of {leveiopmental retardat ton followi~l~ pxrrratal hyperthermra. Interpretation ot the way in whr& hear stress tnl’luenccd SDR learning ability is not undcrst~~~~(I.SeVeral factors that may have affected learning performance were ruled out It is possible that inferior learnmg shility might simply br> a neural &ficita. For example, product of quantitative studies havr demonstrated that normal mace with high brarnwcights show superior learning performance iu those selected for low brainwelg~~ts [ 22. 23, 5 1, s.?]. E?cpcrimental increase of rat bram ceil numbers with prenatal maternal growth hormone administration ts asSo&tad With superior learning ability [ 4.5 1. In h~jn~~s, a head oircumfkrence below - 7, standard deviations for the age is mvariably associated with mental retardation and body hqht and weight below the expected norm 1401. These studies demonstrate that brain size and cell numbers have important behavioral consequences, but whether learning or intellectual deficits associated with brainw~~ght deficits are

PRENATAL

HEAT

STRESS

ON

BRAIN

GROWTH

AND

causally related remains to be demonstrated. The basis for the correlation is not clear. Impaired learning performance in prenatally stressed animals may also have been affected by indirect factors. Thus, heat stress may have affected the nervous and other related systems to cause changes in motivational and/or incentive levels, activity, emotionality or timidity and possibly impulsivity. Although these factors are known to affect learning performance, the majority of studies investi-

149

LEARNING

gating brain-behavior relationships show little appreciation of this problem. This may in part be due to our limited understanding of brain-behavior interactions and to the formidable difficulties in controlling for their confounding influences. A few studies that have made promising attempts in controlling for certain of these influences [ 12, 26, 27, 281, has prompted further research along these lines with heat stressed progeny [ 381.

REFERENCES 1.

2. 3. 4.

5.

6. I. 8. 9.

10.

11. 12. 13. 14. 15.

16.

17.

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