Prenatal morphine exposure attenuates the maintenance of late LTP in lateral perforant path projections to the dentate gyrus and the CA3 region in vivo.

Previously we reported that prenatal exposure to morphine twice daily during gestation decreases proenkephalin levels in adult progeny within the brain, including the dentate gyrus, and alters mu and delta opioid receptors in the hippocampal CA3 region. The lateral aspect of the perforant path contains and releases enkephalin-derived opioid peptides, and induction of long-term potentiation (LTP) in lateral perforant path projections to both the dentate gyrus and the hippocampal CA3 region is blocked by antagonists of opioid receptors. Thus LTP induction at these synapses involves opioid receptor activation mediated by the release of proenkephalin-derived opioid peptides with lateral perforant path activation. Here we show in adult behaving animals, neither LTP induction nor the early phase of LTP (E-LTP) maintenance is altered by prenatal morphine exposure in the lateral perforant path projections to the dentate gyrus and the CA3 region. However, maintenance and longevity of late LTP (L-LTP), as reflected in the magnitude of LTP over days, was attenuated in animals prenatally exposed to morphine. In contrast, in medial perforant path projections to the dentate gyrus and CA3 region, both LTP induction and the maintenance of E- and L-LTP were unaffected by prenatal morphine treatment. Thus a brief prenatal exposure to the opiate morphine produces sustained, and possibly permanent, alterations in L-LTP in the opioidergic lateral perforant path projection. This suggests that prenatal morphine exposure disrupts LTP via disruption of opioid mechanisms involved in LTP maintenance or via disruption of opioid receptor activation during LTP induction, which can subsequently alter LTP maintenance.

Foster mother care but not prenatal morphine exposure enhances cocaine self-administration in young adult male and female rats.

The present study was designed to investigate cocaine self-administration in adult male and female rats exposed prenatally to morphine. Pregnant dams were injected two times a day with either saline, analgesic doses of morphine or no drug at all (controls) on gestation Days 11-18. One day after birth, litters were cross-fostered such that control dams were paired with one another and their litters were crossed; saline- and morphine-treated dams were paired and half of each saline litter was crossed with half of each morphine litter. Thus, each mother (control, saline, and morphine) raised half of her own and half of the adopted litter. At the age of 60 days, males and females were trained first to lever press for sucrose pellets and then for cocaine. Once the lever-pressing behavior was learned and baseline level of this activity was established, animals received a cocaine (.5 mg/kg per infusion) reward for each correct response on the active lever during the next 9-day session. The data demonstrate that adult control, saline- and morphine-exposed male rats self-administer cocaine at a similar rate independent of their prenatal treatment. Adult female rats self-administer cocaine at a higher rate than male rats. Further, saline- and morphine-exposed females in diestrus self-administer more than females in proestrus phase of the estrous cycle, while control females show no such differences. In addition, fostering induces increase in cocaine self-administration in all groups of male rats regardless of prenatal drug exposure. In females, the only fostering-induced increase is in prenatally saline-exposed female rats raised by morphine-treated foster mother. Thus, our results suggest that the prenatal drug exposure does not induce changes in lever-pressing behavior for cocaine reward in adult male and female rats, but it sensitizes the animals to postnatal stimuli such as gonadal hormones and/or rearing conditions that result in increased drug self-administration.

Cross-generational effect of prenatal morphine exposure on neurobehavioral development of rat pups.

Prenatal exposure to opiates can have devastating effects on the development of human fetuses and may induce long-term physical and neurobehavioral changes during postnatal maturation. The present study was aimed at identifying cross-generational effects of prenatal morphine exposure in Sprague-Dawley rats. Pregnant rats were injected subcutaneously with either saline or morphine (10 mg/kg) twice daily during gestational days 11-18. Litter size, percentage of males and females, anogenital distances (AGDs), righting reflex, and body weight were assessed in prenatally morphine-exposed pups (first generation) and their offspring (second generation). Both prenatally morphine-exposed pups and offspring of prenatally morphine-exposed dams exhibited an increased latency to right. Additionally, second generation pups were slower in righting than first generation pups. During the early postnatal period the second generation pups weighed less than the first generation regardless of drug exposure. The AGDs of second generation male pups were decreased relative to the first generation. Our data provide important novel information about the trans-generational effects of maternal opiate abuse that may be useful for understanding/evaluating the teratogenic effects of prenatal opiate exposure.

Field-specific changes in hippocampal opioid mRNA, peptides, and receptors due to prenatal morphine exposure in adult male rats.

Alterations in the opioid system in the hippocampal formation and some of the possible functional consequences were investigated in adult male rats that were prenatally exposed to either saline or morphine (10 mg/kg twice daily on gestational days 11-18). In situ hybridization and Northern blots were used to measure proenkephalin and prodynorphin mRNA, and radioimmunoassays quantified proenkephalin- and prodynorphin-derived peptide levels in the dentate gyrus, CA3, and CA1 subfields of the hippocampal formation. Prenatal morphine exposure in male rats decreases proenkephalin and increases prodynorphin mRNA selectively in the granule cell layer of the dentate gyrus. Similarly, met-enkephalin peptide levels are decreased and dynorphin B peptide levels are increased in the dentate gyrus but not CA3 or CA1 of prenatally morphine-exposed males. In addition, there are decreases in dynorphin-derived peptides in the CA3 subfield. Receptor autoradiography revealed increases in the density of micro but not delta receptor labeling in discrete strata of specific hippocampal subfields in morphine-exposed males. Because alterations in the hippocampal opioid system suggest possible alterations in the excitability of the hippocampal formation, changes in opioid regulation of seizures were examined. Morphine exposure, however, does not alter the latency to onset or number of episodes of wet dog shakes or clonic seizures induced by infusion of 10 nmol [D-Ala2, MePhe4, Gly-ol5]enkephalin into the ventral hippocampal formation. Interestingly, a naloxone (5 mg/kg) injection 30 min before bicuculline administration reverses the increased latency to onset of clonic and tonic-clonic seizures in morphine-exposed males. Thus, the present study suggests that exposure of rats to morphine during early development alters the hippocampal opioid system, suggesting possible consequences for hippocampal-mediated functions.

Expression of proopiomelanocortin and proenkephalin mRNA in sexually dimorphic brain regions are altered in adult male and female rats treated prenatally with morphine.

The present study demonstrates that prenatal morphine exposure on gestation days 11-18 differentially alters proopiomelanocortin (POMC) and proenkephalin (pENK) mRNA in the hypothalamus and limbic system of adult male and female rats. In adult, prenatally morphine-exposed male rats POMC mRNA levels are decreased in the arcuate nucleus of the hypothalamus (ARC), while the pENK mRNA levels are increased in the paraventricular nucleus of the hypothalamus (PVN) and in the ventrolateral subdivision of the ventromedial nucleus of the hypothalamus (VMH), specifically in the ventrolateral subdivision of the VMH. In adult, prenatally morphine-exposed female rats, POMC mRNA levels in the ARC are increased in ovariectomized (OVX) but not in OVX, estradiol benzoate- (EB) or EB- and progesterone- (P) treated females. In contrast, pENK mRNA levels are decreased in the VMH of morphine-exposed, OVX females and increased in EB-treated females. Further, prenatal morphine exposure decreases pENK mRNA in the ARC and increases it in the medial pre-optic area independently of female gonadal hormones. Finally, POMC mRNA levels are increased in the ARC of saline-exposed, EB- or EB- and P-treated females but not in OVX females. Thus, the present study suggests that prenatal morphine exposure sex and brain region specifically alters the level of POMC and pENK mRNA.

Bicuculline seizure susceptibility and nigral GABAA alpha1 receptor mRNA is altered in adult prenatally morphine-exposed females.

Prenatal morphine exposure (5-10 mg/kg twice daily on gestation days 11-18) can adversely affect neurological development, including seizure susceptibility. The present study examines the effects of prenatal morphine exposure on seizure susceptibility to the GABA antagonist and convulsant bicuculline and GABA(A) alpha(1) receptor mRNA in the substantia nigra (SN) of female rats. The results demonstrate that prenatally morphine-exposed ovariectomized (OVX) females and OVX females with estradiol benzoate (EB) replacement have an increased latency to seizure onset compared to controls. In addition, prenatal morphine exposure decreases the area covered by grains of GABA(A) alpha(1) receptor mRNA in the anterior SN in both OVX and EB+progesterone (P)-treated groups, and decreases the number of GABA(A) alpha(1) receptor mRNA-labeled cells/field in EB females. Furthermore, prenatally morphine- and saline-exposed EB and EB+P females had decreased GABA(A) alpha(1) receptor mRNA-labeled cells/field in the anterior SN compared to OVX animals of the same prenatal exposure. These results demonstrate that the long term effects of prenatal morphine exposure in female rats is dependent on their hormonal status, and suggest that seizure susceptibility may be altered via neuropharmacological changes in the GABA system in the SN.

Prenatal morphine exposure decreases susceptibility of adult male rat offspring to bicuculline seizures.

The purpose of this study was to investigate the effect of prenatal exposure to morphine (5-10 mg/kg twice daily on days 11-18 of gestation) on bicuculline seizure susceptibility and to examine the interaction of prenatal morphine exposure and hormonal background in adult male rats. The data demonstrate that prenatal morphine exposure does not affect clonic but decreases susceptibility to tonic-clonic bicuculline seizures in intact male rats. Thus, the present data support our previous work demonstrating alterations in seizure susceptibility of adult morphine-exposed animals.

Prenatal morphine exposure induces age- and sex-dependent changes in seizure susceptibility.

1. Prenatal exposure to morphine induces long-term alterations in seizure susceptibility, which are age-, sex-, and seizure model-specific. 2. Adult male and female rats exposed prenatally to morphine show decreased susceptibility to GABA-regulated seizures. 3. Prenatally morphine-exposed, adult male rats are more sensitive to excitatory amino acid receptor-mediated seizures than control males, control females, or morphine-exposed females.

Prenatal morphine exposure differentially alters learning and memory in male and female rats.

The present study tested the hypothesis that exposure to morphine on prenatal days 11-18 impairs performance on tasks requiring learning and memory in adult male and female rats. In Experiment 1, a symmetrical maze was used to measure learning. In Experiment 2, an eight-arm radial maze was used to assess working spatial memory. The results of Experiment 1 demonstrated that prenatal morphine exposure reduces the time needed to complete the trials, but does not affect the accuracy of performance in male rats. In contrast, prenatal drug treatment had no effects on either the time or the accuracy of performance in female rats. In Experiment 2, both male and female morphine-exposed rats needed more time to complete regular trials (no delay) than controls; however, morphine exposure in male rats did not affect performance on tasks requiring memory, measured with delay trials, but hindered it in ovariohysterectomized (OVX) female rats. In OVX females, replacement injections of both estrogen and progesterone restored the impairment of performance on delay trials produced by prenatal morphine exposure. Thus, the present study demonstrates that prenatal morphine exposure differentially alters performance of adult male and female rats on tasks requiring learning and spatial memory.

Repeated morphine administration during pregnancy attenuates maternal behavior.

The present study tested the hypothesis that repeated administration of morphine on days 11-18 of pregnancy alters maternal behavior. Saline- and morphine-treated mothers were observed with their pups in two experiments. Rats were always tested twice a day during the light and dark phases of the reverse light/dark cycle. In Experiment 1, 12 types of activities and three types of nursing positions of mothers were recorded ten times during each 50-minute session for the 23-day lactation period. A decrease in nursing and active maternal behavior, and an increase in self-care, rearing and sniffing was found in morphine-treated mothers. Additionally, both saline- and morphine-treated mothers exhibited significantly more maternal behavior during the light, and non-maternal activities during the dark sessions of each day. Moreover, both saline- and morphine-treated mothers displayed significantly less maternal behavior and more non-maternal activities as postpartum time progressed. In Experiment 2, a different group of mothers was tested for pup retrieval from postnatal days 1 through 12. Morphine-treated mothers were slower than saline-treated mothers in retrieving all pups into the nest. However, there were no differences in latency to carry the first pup and return him/her to the nest. No unusual maternal behaviors were observed during the retrieval tests. Thus, the present study suggests that morphine administration during the second half of pregnancy attenuates some components of maternal behavior and increases non-maternal activities of mothers.

Prenatal morphine exposure alters estrogen regulation of kappa receptors in the cortex and POA of adult female rats but has no effects on these receptors in adult male rats.

The binding characteristics of kappa receptors were assessed in the frontal cortex (CX), striatum, hypothalamus, preoptic area (POA), cerebellum, and ventral tegmental area of adult male and female rats exposed prenatally to morphine or saline. Prenatal morphine exposure altered estrogen regulation of kappa receptors in the CX and POA of females, but had no effects on kappa receptors in any of the examined brain regions in male rats.

Prenatal morphine exposure alters susceptibility to bicuculline seizures in a sex- and age-specific manner.

Bicuculline was used to investigate seizure susceptibility in pre- and peripubertal male and female rats exposed prenatally to morphine. Morphine-exposed males showed increased seizure susceptibility at prepubertal and decreased susceptibility at peripubertal ages. There was no difference in seizure susceptibility in morphine-exposed females at either age. Therefore, the present data suggest that males are more vulnerable than females to morphine-induced insults during prenatal brain development.

Prenatal morphine exposure enhances seizure susceptibility but suppresses long-term potentiation in the limbic system of adult male rats.

The present study examined the effects of prenatal morphine exposure on NMDA-dependent seizure susceptibility in the entorhinal cortex (EC), and on activity-dependent synaptic plasticity at Schaffer collateral and perforant path synapses in the hippocampus. During perfusion with Mg(2+)-free ACSF, an enhancement of epileptiform discharges was found in the EC of slices from prenatally morphine-exposed male rats. A submaximal tetanic stimulation (2x50 Hz/1 s) in control slices elicited LTP at the Schaffer collateral-CA1 synapses, but neither LTP nor LTD was evoked at the perforant path-DG synapses. In slices from prenatally morphine-exposed adult male rats, long-term potentiation of synaptic transmission was not observed at Schaffer collateral-CA1 synapses, while the submaximal tetanus now elicited frank LTD of synaptic EPSPs at perforant path synapses. These data suggest that prenatal morphine exposure enhances the susceptibility of entorhinal cortex to the induction of epileptiform activity, but shifts long-term plasticity of hippocampal synapses in favor of LTD.

Prenatal morphine exposure differentially alters TH-immunoreactivity in the stress-sensitive brain circuitry of adult male and female rats.

Previously, we demonstrated that exposure to morphine during gestation increases hypothalamic norepinephrine (NE) content and turnover rate in adult male rats and decreases these measures in adult females. To investigate the basis of these alterations, the present study examined the effects of prenatal exposure to morphine on tyrosine hydroxylase immunoreactivity (TH-IR) in the brains of adult male and female progeny. In male rats, prenatal morphine exposure significantly increased the density of TH-IR in cells and fibers in the caudal paraventricular nucleus of the hypothalamus (PVN) and locus coeruleus (LC), but had no effects in the lateral hypothalamus (LH). In female rats that were ovariectomized (OVX), prenatal morphine exposure significantly decreased the density of TH-IR in cells and fibers in the LC. Interestingly, an injection of estrogen in OVX control females reduced the mean optical density of TH-IR in the LC, but it was ineffective in drug-exposed females in the same brain region. Estrogen injections also reduced the mean optical density of TH-IR in the LH but not in the PVN of females, regardless of prenatal drug exposure. Thus, the present study suggests that prenatal morphine exposure induces long-term, sex-specific alterations in TH-IR in the PVN and LC of adult progeny.

Prenatal morphine exposure alters N-methyl-D-aspartate- and kainate-induced seizures in adult male rats.

The purpose of the present study was to investigate whether prenatal exposure to morphine has effects on excitatory amino acid-induced seizures. Adult male rats, exposed on embryonic days 11-18 to saline or morphine, were injected with N-methyl-D-aspartate (NMDA) (150, 175, 200, 225, and 250 mg/kg) or kainic acid (KA) (15 or 20 mg/kg) in adulthood to assess the occurrence and latency to onset of stereotypy and seizures. The latency to onset of stereotypy was significantly increased after 175 mg/kg, and decreased after 200 mg/kg of NMDA in morphine-exposed animals. The lowest dose of NMDA (150 mg/kg) induced seizures in prenatally saline-treated control male rats but not in the morphine-exposed male rats. In the KA-injected group, prenatally morphine-exposed males had shorter latency to onset of wet-dog shakes, but there were no effects on the latency to onset of clonic seizures. The data suggest that prenatal morphine exposure has long-term effects on seizure susceptibility and the onset of stereotypy in the excitatory amino acid-induced seizure models.

Estrogen differentially alters NMDA- and kainate-induced seizures in prenatally morphine- and saline-exposed adult female rats.

The purpose of the present study was to investigate the effects of prenatal exposure to morphine on seizure susceptibility in adult female rats. Adult female rats, exposed to saline or morphine on prenatal days 11-18, were ovariectomized (OVX) and some were injected 48 h prior to seizure testing with estradiol benzoate (EB). To assess the latency to onset of stereotypy and seizures, females received systemic injections of N-methyl-D-aspartate (NMDA; 150, 175, 200 mg/kg) or kainic acid (KA; 10 or 15 mg/kg). Prenatal morphine exposure increased the latency to onset of wet-dog-shakes (WDS) in both OVX and OVX, EB-injected females after the higher dose of KA. However, prenatal morphine exposure increased the latency to onset of stereotypy only in OVX, EB-injected females after the highest dose of NMDA. Prenatal morphine exposure also increased the latency to onset of seizures after the lower dose of KA, but did not change the latency to onset of NMDA-induced seizures. Additionally, an EB injection increased the latency to onset of seizures in both saline- and morphine-exposed females after the lowest dose of NMDA, but decreased the latency to onset of seizures after the lower dose of KA. Thus, the present study demonstrates that prenatal morphine exposure has different effects on the estrogen regulation of the onset of seizures and stereotypy induced by NMDA or KA in adult, OVX female rats.

Prenatal exposure to morphine differentially alters gonadal hormone regulation of delta-opioid receptor binding in male and female rats.

The present study tested the hypothesis that exposure to morphine on gestation days 11-18 differentially alters delta-opioid receptors in the brain of adult male and female rats. In Experiment 1, the binding characteristics of delta-opioid receptors were examined in membrane homogenates from six brain regions, including the hypothalamus (HYP), preoptic area, frontal cortex (CX), ventral tegmental area, striatum (STR) and cerebellum of adult male and female rats. In Experiment 2, the density of delta-opioid receptors was assessed in the CX and STR using receptor autoradiography. Prenatal morphine exposure has no effects on delta-opioid receptors in the brain of gonadally intact, adult male rats regardless of methodology. However, when male rats were gonadectomized in Experiment 2, morphine-exposed males have fewer delta-opioid receptors than controls in the CX but not in the STR. These reductions in cortical delta-opioid receptors are restored by testosterone replacement, demonstrating that prenatal morphine exposure alters testosterone regulation in the CX of male rats. In ovariectomized (OVX) female rats, prenatal morphine exposure increases the density of delta-opioid receptors in the frontal CX. Interestingly, this up-regulation of delta-opioid receptors is not present when the CX is investigated by autoradiography. Moreover, progesterone given alone or in combination with estrogen reduces the density of delta-opioid receptors in the CX and STR of both saline- and morphine-exposed, OVX females. Thus, mid to late gestational morphine exposure differentially alters the influence of adult gonadal hormones on delta-opioid receptors in the CX, decreasing the sensitivity in females and increasing it in males. This is also the first report to demonstrate that gonadal hormones regulate delta receptor densities in brain regions other than the HYP of OVX females.

Prenatal morphine exposure differentially alters seizure susceptibility in developing female rats.

We examined the effect of prenatal morphine exposure (5-10 mg/kg on days 11-18 of gestation) on seizure susceptibility in female rats during development. The effect of morphine exposure on flurothyl-induced seizures was age-dependent. At postnatal day (PN) 15, morphine exposure decreased both clonic and tonic-clonic seizure thresholds compare to saline controls. At PN 25, morphine exposure did not alter the clonic seizure threshold but increased the threshold to tonic-clonic seizure. At PN 38, morphine exposure did not influence either threshold. The data suggest that the effects of prenatal exposure to opioids on seizures are age-related and transient.

Effects of prenatal morphine exposure on rat heterotypical sexual behavior.

Prenatal exposure to morphine inhibits ovarian steroid-dependent lordosis behavior in female rats, and enhances certain components of male sexual behavior in male rats. In the present study, the effects of mid to late gestational morphine exposure on male sexual behavior in females and on female sexual behavior in males were examined in adult offspring. Gonadectomized male rats were injected at weekly intervals with 30 or 60 microg estradiol benzoate and 1.0 mg progesterone and tested for female sexual behavior with stimulus males on 2 consecutive weekly tests. Ovariohysterectomized (OVX) females were injected with 500 microg testosterone propionate (TP) daily for 15 days and tested for male sexual behavior with stimulus females on the last day of TP injection and 1 week later, after TP withdrawal. Prenatal morphine exposure increased the expression of male sexual behaviors in female rats, but it did not increase lordosis behavior in male rats. Thus, exposure to morphine during gestation alters male and female sexual behavior in young adult animals. Because prenatal morphine exposure both defeminized and masculinized adult sexual behavior in female rats, it is possible that female brain development is more vulnerable to prenatal insult such as opiate exposure.