J.M. DiPirro, M.B. Kristal / Brain Research 1014 (2004) 22-33
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proffered the material according to the following schedule:
Days 1-3: 1 g lean ground beef; Day 4: 0.5 g ground
beef + 1 placenta (approximately 0.5 g); and Day 5: 2
placentas. Rats that failed to complete the feeding schedule
successfully were dropped from the study (2 rats of 284
[ < 1%] were excluded).
The habituation period lasted approximately 1 week. The
interval between surgery and testing was therefore 14-18
days.
2.6.2. Testing timeline
Before the start of the test, rats were denied access to
food for a 2-h period to decrease the likelihood that stomach
contents would affect the action of ingested placenta (and
presumably, therefore, POEF). After this period, each rat
was fed 1.0 g of placenta or ground beef control in its home
cage. Ten minutes later, it received an i.c.v. injection of
opioid agonist or vehicle in a separate testing room, and was
placed back in its home cage. Hotplate response latency was
assessed at the time of peak antinociceptive drug effect,
which occurred at 10, 20, and 30 min post-injection for
DPDPE, spiradoline, and DAMGO, respectively. All alge-
siometric tests were conducted in a room other than the one
in which subjects were housed. To minimize circadian
fluctuations in endogenous opioid levels, all testing was
conducted between 0830 and 1130 h, EST.
2.7. Histological examination
At the conclusion of the study, each rat was overdosed
with sodium pentobarbital (0.6 ml, i.p.), and injected though
the i.c.v. cannula with 0.1 Al methyl blue dye. The brain was
then removed, frozen (— 20 °C), and cut into 40-μm
sections. Every 4th or 5th section that showed cannula track
was mounted, stained with Cresyl violet, and saved. Place-
ments were considered to be accurate if: (1) methyl blue dye
was observed in the ventricular system, or (2) the cannula
track could be traced into, but not past, the right lateral
ventricle. In most cases, both criteria were met. Data from
any rat with an inaccurate cannula placement were excluded
from statistical analysis.
2.8. Statistical analysis
2.8.1. Nociceptive threshold
Median tests were used to analyze the differences in
median response latencies between placenta-fed and beef-
fed groups at each dose of each agonist. Nonparametric
analyses were used because the data contained latency scores
that were ‘‘at ceiling’’, (i.e., 55 s), which truncated the
distribution of scores and led to a violation of the assumption
of interval measurement necessary for parametric analysis
[80]. For Experiment 1, in which n < 20 for each comparison,
the probability of the observed values was calculated by the
Fisher exact probability test. For Experiments 2 and 3, in
which n>20 for each comparison, the probability of the
observed values was obtained by chi-square. The alpha level
for all experiments was set at p = 0.05.
2.8.2. Motor integrity
The Fisher exact probability test was used to analyze the
difference in the proportion of rats that exhibited contralat-
eral circling in placenta-fed and control-fed groups at each
dose of the y agonist DPDPE.
3. Experiment 1: D-opioid receptors
The effect of placenta ingestion on the antinociception
produced by the i.c.v. injection of each of five doses of
DPDPE, a y-opioid receptor agonist, was measured.
3.1. Method
3.1.1. Subjects
Seventy-nine virgin female rats, demonstrating normal
estrous cyclicity, were used.
3.1.2. Drug
The y-receptor-selective agonist DPDPE (a gift from
NIDA) was used. DPDPE was chosen because it displays
a binding affinity for the y-opioid receptor that is 100 times
greater than its affinity for ι and n receptors [24,32,61].
DPDPE was dissolved in 0.9% physiological saline and
injected within 1 h of entering solution.
3.1.3. Design
The design of this experiment was a 2 x 5 between-
subjects factorial: Enhancer (1.0 g placenta or 1.0 g beef
control) x Dose of У agonist (0, 30, 50, 62, or 70 nmol
DPDPE in 4.5 ιl, i.c.v.). Rats were randomly assigned to 1
of 10 experimental conditions and tested only once.
3.2. Results and discussion
Of the 79 rats tested in Experiment 1, three rats failed to
eat the proffered enhancer (placenta or beef control) during
testing, and six had inaccurate cannula placements. Only the
data from the remaining 70 rats were used for analysis.
3.2.1. POEF and d-receptor-mediated antinociception
The effect of placenta ingestion (and therefore, presum-
ably POEF ingestion) on y-mediated antinociception is
depicted in Fig. 1. Placenta ingestion significantly enhanced
the antinociception produced by both the 62- and 70-nmol
doses of DPDPE (pV 0.025, median test). At those doses,
placenta-fed rats exhibited response latencies that were
more than 200% of those of their control-fed counterparts
(62 nmol DPDPE: placenta Mdn= 55.0 s, control
Mdn= 13.9 s; 70 nmol DPDPE: placenta Mdn= 55.0 s,
control Mdn= 25.6 s). Placenta ingestion did not alter
response latency in rats injected with vehicle or with the