Physiology & Behavior,Vol. 55, No. 5, pp.

971-974, 1994
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The Interaction Between Prenatal Stress and

Neonatal Handling on Nociceptive Response
Latencies in Male and Female Rats
JAMES W. S M Y T H E , .1 C H E R Y L M. M c C O R M I C K , * ~ " J O S E P H ROCHFORD*
A N D M I C H A E L J. M E A N E Y *

*Developmental Neuroendocrinology Laboratory, Douglas Hospital Research Centre, Department of Psychiatry,

McGill University, 6875 LaSalle Blvd., Montreal, Quebec, Canada H4H 1R3 and tDepartment of Psychology,
Bates College, Lewiston, ME 04240

R e c e i v e d 9 J u l y 1993

SMYTHE, J. W., C. M. McCORMICK, J. ROCHFORD AND M. J. MEANEY. The interaction betweenprenatal stress and
neonatalhandlingon nociceptiveresponselatenciesin maleandfemale rats. PHYSIOL BEHAV $5(5) 971- 974, 1994.--Neonatal
handling produces physiological and behavioral changes that persist into adulthood. These effects are opposite to those resulting
from prenatal stress (PS). We examined the interaction between PS and handling on nociception in adult male and female rats.
Randomly selected pregnant rats were subjected to restraint stress on days 13-17 of gestation for 25 mill each day, or left
undisturbed. At birth, selected stressed/nonstressed litters were assigned to be handled. Handling consisted of 15 rain of separation
from the dam, once per day, from postnatal days 1-14. At 4 months of age, rats were placed on a 50°C hot plate, and their latencies
to paw lick were recorded. Prenatal stress and handling interacted to affect latencies in male rats. Handled (H)/PS rats had
significantly lower paw lick latencies than nonhandled (NH)/PS rats (p < 0.05). However, handling had no effect on the male
offspring of control dams. Handling elevated paw lick latencies in the female offspring of control dams, an effect that was most
pronounced in diestrous vs. estrous rats. The NH/PS rats showed significantly elevated latencies compared to NH/NS rats (p <
0.05). These results suggest that handling effects on nociception are most apparent in rats subjected to PS; in males at least, these
effects would otherwise not be present.

Paw lick Neonatal Handling Prenatal Stress Sex differences Nociception

N E O N A T A L handling has been found to alter behavioral and
endocrine responses to stress. Adult rats, handled (H) as pups,
secrete less adrenocorticotrophin (ACTH) and corticosterone
(CORT) in response to restraint stress compared to nonhandled
(NH) controls. Moreover, hormone levels in H rats return to basal
levels more quickly than in NH animals (1,6,10,11). In addition
to physiological changes brought about by handling, behavioral
alterations have also been reported. Handled rats manifest less
fearfulness than NH rats as measured by their latencies to ap-
proach novel items in an open field (3,4).
Handling also elevates paw lick latencies in adult mice as
assessed by the tail flick task, and augments morphine-induced
hypoalgesia assessed by both tall flick and hot plate tasks (2,17).
These data suggest that handling exerts enduring influences on
antinociceptive systems, perhaps occurring concomitantly with
alterations in hypothalamic-pituitary-adrenal (HPA) function.
The effects of handling on HPA function and behavior are
opposite in some respects to those produced by prenatal stress
(PS). Prenatally stressed rats under 21 days of age exhibit ele-
vated A C T H and C O R T secretion in response to foot shock
stress, an effect that apparently disappears by adulthood (20,21).
Effects of PS on HPA responses to stress are sex dependent.
McCormick et al. [(9), submitted] reported that PS increased
plasma C O R T and A C T H responses to restraint stress in adult
female rats, but not in males. Behaviorally, PS is associated with
exaggerated fearfulness in adult rats as measured by increased
freezing, poor plus-maze performance, increased levels of ultra-
sonic vocalizations, elevated defecation, and reduced open field
ambulation (5,12,19-21).
Given the contrasting behavioral and physiological responses
of rats to handling and to PS, we wondered whether handling
might attenuate the effects of PS. Indeed, Wakshlak and Wein-

' Requests for reprints should be addressed to James W. Smythe, Ph.D., Douglas Hospital Research Center, 6875 LaSaile Blvd., Montreal, Quebec,
Canada H4H 1R3.

971
972 SMYTHE ET AL.

stock (22) reported that handling elevated ambulation scores in returned to their home cages. A limit of 90 s was arbitrarily
prenatally stressed rats, that ordinarily show little open field ac- placed on each rat to reduce discomfort to the animals. Testing
tivity. Moreover, the elevated defecation observed in prenatally took place between 1200 and 1500 h. Because estrogen and pro-
stressed male rats was reduced by handling, and anxiety exhibited gesterone affect nociceptive thresholds (18), vaginal smears were
by female rats in response to PS was similarly diminished. To taken from female rats prior to testing to assess estrous cycle
our knowledge, modulation of the effects of PS by handling on status. Females were characterized as being in either estrous (es-
other behavioral measures has not been investigated. Kinsley, trous and metestrous) or diestrous (diestrous and proestrous)
Mann, and Bridges (8) found that PS reduced morphine analgesia groups, and were analysed separately. We observed no overt dif-
in male rats, but enhanced it in female rats. Basal pain thresholds ferences in estrous cycle across the various treatment conditions.
were lower in prenatally stressed females compared to non-
stressed controls. In contrast, there were no differences between Statistical Analysis
control and prenatally stressed males. Furthermore, Insel et al.
Hot plate response latencies (paw lick) were subjected to a
(7) demonstrated that PS results in lowered striatal densities of
PS x handling factorial ANOVA. Data for males and females
mu opiate receptors. The impetus for the present study was to
were analysed separately to facilitate interpretation. All post hoc
determine if handling and PS would interact to affect basal no-
analyses were performed using Newman-Keuls procedure (al-
ciception as measured by paw lick responses to a hot plate. Our
pha = 0.05).
objectives were to: 1) determine the effects of handling on no-
ciceptive thresholds in both males and females; and 2) examine
RESULTS
the interaction between PS and handling on pain thresholds.
Basal Hot Plate Nociception
METHOD
Data for males and females were analysed separately because
Subjects of the effects of estrous cycle on hot plate responses. A PS x
handling ANOVA for the males revealed no main effects of PS
The offspring of Long Evans Hooded rats served as subjects
or handling; however, the interaction term was significant,/7(1,
in the study. Dams were purchased at 10 days of gestation from
56) = 4.13, p < 0.04. As is evident from Fig. 1, handling had
a local supplier (Charles River Canada, St. Constant, Quebec),
no influence on nociceptive thresholds in nonstressed rats; how-
and were acclimatised for 3 days before being subjected to stress
ever, I-I/PS rats had significantly lower paw lick latencies than
(see below). Dams and their pups were housed in polycarbonate
NH/PS rats (/9 < 0.05). Thus, PS alone had no influence on
maternity cages, with wood chip bedding. Lab chow (Purina) and
nociception in male rats. Such differences were only manifested
water were provided ad lib. The day of birth was designated
if handling was superimposed in addition to PS.
postnatal day 1. Litters were not culled, although stillborn pups
The results for the females are shown in Fig. 2. Rats were
were removed. There were no group differences in litter size,
categorized as being either in estrus or diestrus. A three-way
number of stillborns, or in sex ratios across treatments. Following
ANOVA (cycle × PS x handling) revealed a significant main
birth, litters were left undisturbed for 2 weeks, except for han-
effect of estrous cycle, F(1, 56) = 4.03, p < 0.05, and a signif-
dling (see below). At 21-23 days of age, pups were weaned to
icant main effect of PS, F(1, 56) = 10.62, p < 0.003. There was
hanging colony cages and housed in groups of five (same sex also a significant interaction between PS and handling, F(1, 56)
only). Animals were maintained under normal light cycle con-
= 5.28, p < 0.03. In nonstressed rats, handling elevated noci-
ditions (on at 0800 h; off at 2000 h).
ceptive thresholds, an effect that was far more pronounced in
diestrous females as opposed to estrous females (p < 0.05). Pre-
Treatmen~ natal stress markedly elevated nociceptive thresholds in NH rats
Prenatal stress procedure. On days 13-17, a group of ran- (p < 0.05) compared to nonstressed, NH rats.
domly selected dams was placed into Plexiglas restrainers
(Fischer Scientific, Montreal, Quebec) for a period of 25 min, DISCUSSION
once per day. This procedure was always done at 1400 h. All
The present study demonstrates that the effects of postnatal
other dams were left undisturbed in their cages.
environmental events are influenced by both the prenatal envi-
Neonatal handling procedure. Nonstressed and prenatally ronment and the gender of the animal. Paw lick latencies of male
stressed rat pups were randomly assigned to the H or NH con-
ditions. The handling procedure was the same as that used in our
previous studies (10,11). From postnatal days 1 through 14, dams
of H pups were removed from their home cages and placed in
clean, empty cages. Pups were then removed and placed in sep-
:°] • NS
arate maternity cages, lined with paper towel. After 15-20 rain, IPS
pups were returned to their home cages, followed shortly there-
after by the dams. Handling always took place at approximately
1500-1600 h. The NH pups and their dams were undisturbed
during this 14-day period. Cage maintenance was not undertaken
for any group until day 14.

Procedure J

Nociceptive responses were assessed at 4 months of age. Rats

Handled Non-Handled
were taken from their home cages and placed onto a heated steel
plate (50°C). An experimenter who was blind to group member- FIG. 1. The effects of prenatal stress and handling on paw lick latencies
ship measured the latency to paw lick (usually the hindpaw). Rats in male rats. Means _ SEM are shown, n --- 9-16/group. *p < 0.05
were then immediately removed from the hot plate apparatus and relative to the NS/NH group.
P R E N A T A L STRESS A N D N E O N A T A L H A N D L I N G 973

DIESTROUS nonstressed rats, handling elevated nociceptive thresholds, an ef-

fect that was greater in diestrous animals. Prenatal stress by itself
q- dramatically elevated nociceptive thresholds in NH diestrous
• NS
rats, an effect that was less pronounced in NH estrous rats. Ratka
and Simpkins (18) have shown that estrogen treatment of ovari-
,d lIPS
ectomized rats produces hyperalgesia on basal responding to a
~ so
m hot plate. Our results are consistent with this finding.
The present data also support the notion that handling can
2 attenuate some of the consequences of PS. Even though both
~. 311
handling and PS elevate nociceptive thresholds (PS more so than
.~ 2o handling), handling blocked the large increase in nociceptive
~ 1o thresholds observed in prenatally stressed, H female rats. Thus,
handling elevates nociceptive thresholds in nonstressed rats, but
o
Handled Non-Handled also reduces the PS-induced elevation in nociceptive thresholds.
Kinsley et al. (8) reported that PS lowered basal pain thresholds
in female rats, whereas our results are the exact opposite of those
ESTROUS findings. They used a tail flick task, but in other respects our
procedures were similar (e.g., PS in both studies was done in the
third trimester; only diestrous rats were used). The source of this
** • NS
discrepancy is not clear.
Neonatal handling also has consequences for behavioral ex-
~ • Ps
- s01 pression, but clearly not always in opposition to those of PS, nor
eL 40 is handling always able to supersede the effects of PS. Peters
(15,16) has reported that 5-hydroxytryptamine (5-HT) turnover
2
311 is elevated in the brains of neonatal rats born to prenatally
II
~ 2o stressed dams. Neonatal handling also elevates 5-HT turnover in
7-day-old rat pups (14). Thus, PS and postnatal handling may
have some common neural targets, although the behavioral con-
o sequences of both are different. At this point in time, we cannot
Handled Non-Handled say what mechanism is responsible for the results obtained in the
FIG. 2. The effects of prenatal stress and handling on paw lick latencies present study. Nevertheless, it is interesting that PS and handling
in female rats. The top panel shows rats that were categorized as being had such opposite effects because it suggests that handling is not
in diestrus at the time of testing, and the bottom panel shows data from necessarily a stressor, at least not in the same way that PS is.
estrous rats. Values shown are means _+ SEM. n = 5-9/group. *p < Either the consequences of third trimester PS and handling stress
0.05 relative to the NS/NH group; are markedly different due to the animal's maturational state, or
**p < 0.05 relative to the NS/NH group. postnatal handling is not a stressor.
In summary, the behavioral effects of PS and handling are
different. Males display no handling effect in the absence of pre-
rats did not vary between the H/NH nonstressed groups. How-
natal stress and females exhibit a pronounced handling effect that
ever, in PS animals, handling significantly reduced paw lick la-
changes direction following PS. The mechanisms underlying
tencies. Thus, an animal's sensitivity to environmental manipu-
these changes are unknown.
lations (e.g., handling) may be heightened by PS. Therefore, the
magnitude of a handling effect may vary in studies where the
prenatal environment is not always the same. This may have
ACKNOWLEDGEMENTS
importance for researchers interested in handling effects: such H/
NH differences may only be expressed if the animals are sub-
This research was supported by a Medical Research Council of Can-
jected to some degree of PS. In the current study, handling had ada grant to M.J.M. and a Natural Sciences and Engineering Research
no measurable effect on male rats, but combined with PS, their Council of Canada grant to J.R.J.W.S. and C.M.M. were supported by
heightened sensitivity to handling was apparent. Natural Sciences and Engineering Research Council of Canada Postdoc-
Our findings for female rats demonstrate that the estrous phase toral Fellowships. M.J.M. holds an MRC Scientist Award and J.R. is a
of the estrous cycle lowers paw lick latencies in all animals. In chercheur boursier of the Funds de la Recherche en Sante du Quebec.

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