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0-80 stainless-steel screws. At the end of surgery, a stain-
less-steel obturator, cut flush with the guide cannula, was
inserted into the guide cannula. The internal cannula, used
to deliver drug to the lateral ventricle, was cut 1 mm longer
than the tip of the guide cannula, so that the site of drug
injection would be beyond any scar-tissue formation or
gliosis that may have occurred between surgery and testing.
2.3. Drug injections
Intracerebroventricular drugs were injected with a Har-
vard microinfusion pump (Model 944) at a rate of approx-
imately 1 Al/min. The volume ranged from 4.0 to 4.5 Al, but
remained constant within a particular experiment. During
injection, the rat was handheld. After injection, the internal
cannula was left in position in the ventricle for a period of
30 s to allow for drug dispersal from the cannula tip. The
internal cannula was then replaced with the obturator in
order to minimize backflow of drug into the guide cannula.
Each rat was tested with only one opioid agonist, although
cross-tolerance to different opioid receptor-preferential ago-
nists is minimal [69,99], to avoid the possibility of a
repeated-testing effect. Agonist doses were determined in
pilot studies by their ability to generate a small amount of
antinociception (10 - 40% increase from baseline response
latency) so that POEF-induced effects, if present, could be
distinguished from simple drug-induced effects, within the
55-s test.
2.4. Placenta collection
Placenta was harvested on Day 21 of pregnancy (pres-
ence of sperm = Day 1) from females killed with CO2.
Placenta was immediately frozen (— 40 t>C) and stored for
later use. During testing, frozen placenta was warmed for 15
min to 37 jC in a heating block, weighed, and immediately
presented to the subjects. This procedure is standard for our
laboratory (e.g., Ref. [43]).
2.5. Behavior assays
2.5.1. Antinociception assay
The detection of antinociception produced by opioid
receptor-selective agonists depends on the type of antinoci-
ception test and the route of drug administration. The
present study used a hotplate (Life Science Instruments,
model 39D), set at 52 jC, to measure nociceptive threshold,
because this particular assay is sensitive to the antinocicep-
tion induced by i.c.v. injection of all three of the opioid
agonists used. The hotplate assay was adapted from that
originally described by Woolfe and McDonald [97]. A
rectangular Plexiglas chamber (28 cm high) with removable
top was used to confine the rats to a 28.8 x 26.6 cm hotplate
surface during testing. Nociception (pain or discomfort)
threshold was quantified as latency (in seconds) to lick the
hindpaw or jump vertically (all paws simultaneously leaving
the plate surface) after placement of the rat on the hotplate.
Immediately after nociceptive threshold (response latency)
determination, rats were removed from the hotplate. If no
response was observed, hotplate exposure was terminated at
55 s in order to avoid tissue damage. Each rat was tested
only once so that learning effects would not influence
hotplate performance. All testing was performed by a tester
who was blind to experimental conditions.
2.5.2. Motor activity assay
The behavioral effects of opioids are not limited to
antinociception, and include a pronounced effect on motor
activity, which might confound pain-threshold determina-
tion in a hotplate assay. This motor effect varies as a
function of opioid receptor type and the degree of activation
(i.e., dose and type of agonist used to stimulate the recep-
tors). Pilot data for the present study indicated that no
apparent motor effects were produced by agonist injection,
except in placenta-fed rats treated with the highest dose of
the y agonist (Experiment 1); those rats displayed circling
contralateral to the site of drug injection. In order to separate
any treatment-induced motor effects from antinociceptive
effects, if possible, circling and nociceptive threshold were
measured in the same rat for all rats in Experiment 1. Also,
an additional (i.e., 5th) dose of the y agonist, slightly lower
than the highest dose, was tested to dissociate y antinoci-
ception from circling. Therefore, to assess y receptor motor
effects, the presence or absence of contralateral circling
(tight, head-to-tail turns) was determined for all rats given y
agonist injections. Circling was assessed at the time of the
hotplate test (i.e., 10 min after agonist injection).
2.6. Testing procedures
2.6.1. Pretesting procedures
To minimize stress (and endogenous opioid effects)
during testing, each rat was given a postsurgical recovery
period of 7 - 10 days before being habituated to all testing
procedures: (1) experimenter (each rat was handheld for 3 -
5 min/day for 7 days); (2) removal of obturator (cannula was
unscrewed 1 time/day for 7 days); (3) hotplate (rats were
exposed to room-temperature hotplate for 5 min/day for 2
days); and (4) feeding procedures (fed testing substance
once/day for 5 days).
Habituation to feeding procedures over a 5-day period
was designed to ensure that subjects would reliably eat
placenta or control substance (ground beef) within the 5-min
access, because very few virgin Long - Evans rats are
spontaneous placentophages and many exhibit neophobic
reactions to novel food [38 - 40]. During this time, once per
day, each rat was proffered 1 g of novel food (ground beef,
placenta, or a mix), presented in a glass dish (2.5 cm
diameter), in addition to standard rat chow. The novel food
remained in the cage until it was eaten; if this did not occur
within the first 1 - 2 h of presentation, rat chow was then
removed until the novel food was eaten. Each rat was