HYPOTHALAMIC CUTS AND MATERNAL BEHAVIOR
631
either eat donor placenta when first exposed to it, or they
avoid it (for review, see [22])—knife cuts were made in rats
that were spontaneously attracted to placenta as virgins
(placentophages), as well as in those that avoided it
(nonplacentophages).
Experiment 2 was conducted to determine the influence
of parturitional experience on the behavioral effects ob-
served in Experiment I.
EXPERIMENT 1
METHOD
Subjects
Subject rats (n=45), pup-donors, and placenta-donors
were Long-Evans females, 3-6 months old, born and raised
in our laboratory. Their parents had been purchased from the
Charles River Breeding Laboratories. Each rat was housed
individually in a 24×19×18-cm wire-mesh cage with food
(Charles River Rat/Mouse/Hamster Formula 3000) and water
available ad lib except as indicated below. All rats were
maintained on a 14 hr on/10 hr off light cycle with the on
phase beginning at 0600 hr (EST).
Procedure
Placentophagia pretest. Daily vaginal smears were ob-
tained from each rat, beginning at about 60 days of age. After
verification of normal estrous cyclicity, the rat was given a
placentophagia pretest. Donor placentas were obtained sur-
gically from CO2-killed, Day 21 pregnant females, then
placed in small plastic vials with a few drops of physiological
saline and frozen at —20oC until needed. A few minutes be-
fore presentation, the placentas were thawed and warmed to
,about 30oC, then placed individually into glass dishes and
presented for 15 min to the rats, which had been placed in
wire-mesh cages in a quiet room 2 hr earlier [23]. After the
subject’s response to placenta was recorded, it was returned
to its home cage. The testing procedure was terminated
when the rat ate placenta and was classified as a placen-
tophage, or after refusal to eat placenta in three consecutive
daily tests, in which case she was classified as a nonplacen-
tophage. Previous research has indicated that once a virgin
eats placenta, she is likely to eat it whenever it is made
available subsequently; virgins that refuse to eat on all three
test days are not likely to eat placenta on subsequent occa-
sions except during delivery or unless coerced by food dep-
rivation [23].
Maternal-behavior pretest. One to two weeks after the
placentophagia pretest, each rat was habituated overnight to
a 45 × 19×25-cm plastic cage containing 3 cm of coarse saw-
dust and fitted with a standard wire-grill top containing food
and water. The next day, four foster pups, 3-8 days of age,
were scattered about the floor of the cage, and the rat’s
responses during the next 15 min were noted. A rat was
excluded from the experiment if she retrieved, attacked, or
ate any of the pups. Spontaneous retrievers and cannibals
constitute a very small proportion of our rat colony; because
such animals are identified in numbers too low to be distrib-
uted evenly across groups, they were instead excluded from
the experiment. At the end of this test the pups were re-
turned to their mother and the test rat was returned to its
home cage.
Confirmation of nonplacentophage status. Each rat in-
cluded as a nonplacentophage in this experiment was re-
quired to demonstrate a refusal to eat placenta in a fourth
test conducted as described in the placentophagia pretest.
This Lday test occurred on a non-proestrus day of the es-
trous cycle 1 to 2 weeks prior to time-breeding.
Knife-cut surgery. Bilateral knife cuts were made
Stereotaxically in rats anesthetized with methoxyflurane
(Metofane, Pitman-Moore) on the afternoon of Day 20 of
pregnancy. The knife was a modified version of that de-
scribed by Sclafani and Grossman [53] and consisted of a
28-ga guide cannula bent and filed at one end in such a way
that a 38-ga stainless steel wire forced through the cannula
extended in the direction of the curved end. For MPO cuts
the cannula was inserted at 2.2 mm anterior to bregma and
1.3 mm lateral to midline, and lowered until the bottom of the
wire would, when extended, reach 7.0 mm below the skull
surface [47]. The wire was then forced through the cannula
and out the curved end in the sagittal plane to a length of
approximately 1.2 mm from the cannula tip. The knife cut
was made by lowering the cannula with the extended wire a
distance of 2.0 mm. The cannula was then raised to 7.0 mm
below the skull surface, the wire was retracted, and the can-
nula was withdrawn from the brain. This procedure was then
repeated on the other side of the brain. MFB cuts were made
by inserting the cannula 1.2 mm posterior to bregma and 2.2
mm lateral to midline, lowering it to 8.0 mm below the skull
surface, extending the inner wire approximately 1.5 mm
toward midline, lowering the knife assembly to 9.0 mm
below the skull surface, raising it to the initial point of ex-
tension, retracting the wire, and withdrawing the cannula
from the brain. The procedure was then repeated on the
other side of the brain. Control rats receiving MPO or MFB
sham cuts were subjected to the same surgical treatments
described above, except that the inner wire was not ex-
truded. Each rat in the ASYM Group received a unilateral
MPO cut and a contralateral MFB cut. Half of the rats in the
ASYM-Control Group received a unilateral MPO cut and a
sham MFB cut, and the other half received a unilateral MFB
cut and a sham MPO cut.
Measures of postoperative food and water intake. After
the surgery, each rat was weighed and placed in a wire-mesh
cage containing measured amounts of food and water.
Twenty-four hours later, on Day 21, body weight and food
and water intakes were determined. Debilitated rats were
excluded from further experimentation. Healthy subjects
were moved to plastic cages containing a small amount of
sawdust, a supply of paper strips, and food and water. The
cages were placed in a room separate from the main colony
that was equipped with a remotely controlled low-light video
camera.
Placentophagia. maternal-behavior, and nestbuilding
observations and testing. Beginning on Day 22 of pregnancy
each rat was observed for parturition during frequent spot
checks. At delivery, continuous observations were ,made to
determine the incidence of placentophagia, the incidence of
cannibalism, and the condition and number of pups. The
time of onset and duration of parturition were also noted.
One hour after completion of delivery, each mother was pre-
sented for 15 min with 5 donor placentas in a glass dish.
Six hours after completion of delivery, each mother was
weighed, the number of delivered placentas that remained
uneaten was noted, the litter and remaining placentas were
removed, and the average pup weight was determined. Then
4 foster pups, 3-8 days old, were scattered about the cage,
and during the next 15 min retrieval, pup licking, and crouch-