J.M. DiPirro, M.B. Kristal / Brain Research 1014 (2004) 22-33
23
including those induced by the physiology of late preg-
nancy, peripheral or central injection of morphine, vagi-
nal/cervical stimulation, footshock, or endogenous opioid
release [19,20,42,45,87]. POEF does not, however,
modify antinociception that results from the non-opioid
analgesics aspirin [44] or nicotine [73]. Injection of
opioid receptor antagonists such as naloxone or naltrex-
one blocks POEF enhancement, presumably by removing
the opioid activity upon which POEF acts [41]. These
data suggest that the modulatory influence of POEF
ingestion on pain suppression is limited to opioid-
mediated mechanisms. However, the effects of POEF do
not extend equally to all opioid-mediated phenomena.
POEF ingestion does not affect morphine-mediated
hyperthermia [1], and it suppresses contralateral circling
produced by unilateral morphine injection into the ventral
tegmental area (VTA) [90].
The different effects of POEF action on different
opioid-induced phenomena are most likely mediated by
different opioid receptor systems (receptor specificity), at
different anatomical sites (location specificity), or both.
Previous work has shown that POEF acts on the central,
rather than peripheral action of morphine [19], but it does
not appear to work directly in the CNS. Gastric vagotomy
blocks the effect of ingested afterbirth material on mor-
phine antinociception [87], and this block seems to be due
to the disruption of gastric vagal afferent fibers, rather
than to the disruption of the efferent fibers that influence
digestion [72]. POEF ingestion enhances antinociception
produced by morphine [1,12,19,20,43,88], a nonselective
A-opioid agonist that has activity at all of the opioid
receptors (i.e., AHy>n) [25]. The strategic location of
A-, y-, and n-opioid receptors at different points of the
opioid-antinociception system in brain and spinal cord
[50,51], where each is involved in the mediation of
antinociception [4,6,9,13,27,30,31,66,82,96], makes each
receptor type a potential candidate for involvement in the
POEF effect. Direct tests of receptor specificity, however,
have yet to be performed.
The present series of experiments was designed to
examine the contribution of each receptor separately by
investigating the modulatory influence of ingested POEF on
antinociception produced by independent central pharmaco-
logical activation of each of the three principal opioid
receptor types. In each of the three experiments, placenta
ingestion was combined with the intracerebroventricular
(i.c.v.) injection of one of three different opioid receptor
selective agonists: y-preferring [D-Pen2,D-Pen5]enkephalin
(DPDPE) [24,32,61]; A-preferring [D-Ala2,N-MePhe4,Gly5-
ol]enkephalin (DAMGO) [24,28,32]; or n-preferring spira-
doline [46,93]. In the present study, we hypothesized that
placenta ingestion would enhance antinociception selective-
ly induced at the y-opioid receptor by DPDPE injection
(Experiment 1), the A-opioid receptor by DAMGO injection
(Experiment 2), and the n-opioid receptor by spiradoline
injection (Experiment 3).
2. Materials and methods: general
2.1. Subjects
Subjects were 282 experimentally naive, virgin female
Long-Evans (hooded, Blue Spruce) rats, 3-5 months old,
weighing 250-350 g. All subjects were born and raised in
our laboratory in the Psychology Department’s Behavioral
Neuroscience Complex at the University at Buffalo and
were the first- or second-generation offspring of rats pur-
chased from Harlan Sprague Dawley. All procedures were
approved by the University at Buffalo Institutional Animal
Care and Use Committee.
Rats were maintained in a controlled environment with
an ambient temperature of 22 F 1 jC, a relative humidity of
40 - 60%, and a 14-h on/10-h off light - dark cycle (lights on
at 0500 h, EST). Rats were housed individually in
32 x 20 x 20 cm, standing, clear plastic cages, and were
allowed ad lib access to food (Agway Prolab Rat/Mouse/
Hamster Formula 3000) and water, except where otherwise
stated.
From the age of 2 months, each rat was monitored daily
for estrous cycle stage by vaginal smear; rats were consid-
ered to be reproductively mature when they exhibited
normal estrous cyclicity (i.e., two consecutive cycles of
4 - 5 days). After reaching maturity, each rat underwent
stereotaxic cannula implantation.
2.2. Stereotaxic surgery
All rats received a single, permanent, indwelling guide
cannula through which opioid agonists could be injected
directly into the right lateral ventricle. In Experiments 1 and
2, surgery was performed while rats were anesthetized with
sodium pentobarbital (40 mg/kg, i.p.) after they had been
food deprived for 8 h. Atropine sulfate (4 mg/kg, s.c.) to
suppress mucus secretion was administered shortly after the
sodium pentobarbital had taken effect. In Experiment 3, rats
were anesthetized with ketamine hydrochloride (21.8 mg/
kg, i.p.) and xylazine (26 mg/kg, i.p.) because sodium
pentobarbital was no longer available. Rats that were
anesthetized with ketamine - xylazine (Experiment 3) were
not food-deprived during the pre-surgery period. All rats
were injected with Combiotic (0.05 ml, i.m.—Experiments
1 and 2) or Baytril (0.04 ml, i.m.—Experiment 3) to prevent
infection.
During surgery, rats were secured in a Kopf stereotaxic
apparatus and permanently implanted with a 22-ga stainless-
steel cannula (Plastic Products) inserted into the right lateral
ventricle at the following coordinates: A-P= 0.0 mm (breg-
ma); L = — 2.0 mm (center of midsagittal sinus); D-
V = — 2.8 mm (measured from dura), with the incisor bar
positioned 5 mm above the interaural line. The coordinates
were modified from the stereotaxic atlas of Pellegrino,
Pellegrino, and Cushman [67]. The guide cannula was
anchored to the skull with dental cement affixed to three