Romana Šlamberová

Age-specific N-methyl-D-aspartate-induced seizures: perspectives for the West syndrome model.

Summary: Purpose: With intraperitoneal N‐methyl‐D‐aspartate (NMDA; 15–200 mg/kg) administration, we attempted to develop an animal model of age‐specific West syndrome to serve for testing of putative anticonvulsant drugs and to determine the mechanisms of this disorder.

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 exposure to morphine differentially alters gonadal hormone regulation of δ-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.

Autoradiographic evidence that prenatal morphine exposure sex-dependently alters μ-opioid receptor densities in brain regions that are involved in the control of drug abuse and other motivated behaviors

The present study examined the effects of prenatal morphine exposure on mu-opioid receptor density in young adult male and female rats to assess the long-term alterations in several brain areas including the nucleus accumbens (NAc), bed nucleus of stria terminalis (BNST), and the basolateral (BLA), lateral (LA), central (CeA), and posteromedial cortical (PMCoA) amygdaloid nuclei. These brain areas are involved in motivating and rewarding behaviors of opiates and other drugs of abuse. The reinforcing actions of opiates appear to be mu-opioid receptor dependent. The results demonstrate that in male rats, prenatal morphine exposure significantly increases the density of mu-opioid receptors in the NAc and PMCoA. In contrast, the same prenatal morphine exposure reduces the density of mu-opioid receptors in the BLA, while increasing it in the CeA and without effects in the LA or BNST. In female rats, prenatal morphine exposure has no effects on the density of mu-opioid receptors in the above six brain areas, but the density of these receptors is dependent on the presence or absence of ovarian hormones. Thus, the present study demonstrates that mid- to late gestational morphine exposure induces long-term, sex-specific alterations in the density of mu-opioid receptors in the NAc and amygdala. Moreover, this prenatal morphine exposure also eliminates sex differences in the density of mu-opioid receptors in the NAc, CeA, and PMCoA but not in the BLA, LA, and BNST.

Adrenocorticotropin Stress Response but Not Glucocorticoid-Negative Feedback Is Altered by Prenatal Morphine Exposure in Adult Male Rats

The present study was designed to investigate the effects of prenatal morphine exposure on the hypothalamic-pituitary-adrenal (HPA) axis-regulated stress responses by measuring restraint stress-induced changes in the adrenocorticotropic hormone (ACTH) and corticosterone (CORT) levels. In experiment 1, plasma levels of ACTH and CORT in prenatally morphine-, saline-exposed and control male rats were determined before and at several times after restraint stress. There were no statistically significant differences in plasma ACTH and CORT levels before restraint stress between the groups. However, prenatal morphine exposure dampened the stress-induced increase and spontaneous recovery of ACTH levels after the restraint stress. There were no differences in plasma CORT levels between the three groups either before or at any time after restraint stress. Experiment 2 was designed to investigate the sensitivity of negative feedback of glucocorticoids using the dexamethasone (DEX) suppression test. DEX was administered at different doses (0.001, 0.01, 0.1 and 1.0 mg/kg) and ACTH and CORT plasma levels were measured before and at several times after restraint stress in prenatally morphine- and saline-exposed males. DEX pretreatment eliminated the differences observed in ACTH responses to stress in morphine- and saline-exposed males. DEX pretreatment dose dependently suppressed the restraint stress-induced increased plasma ACTH concentration. In plasma CORT levels, DEX pretreatment dose dependently suppressed the restraint stress-induced increased plasma CORT concentration regardless of prenatal drug exposure. Thus, the present study demonstrates that prenatal morphine exposure alters the ACTH and CORT responses to stress but not the sensitivity of negative feedback of glucocorticoids.

Impact of maternal morphine and saline injections on behavioral responses to a cold water stressor in adult male and female progeny.

The purpose of the present study was to test the effects of maternal morphine and saline injections on chronic cold water stress responses in three groups of adult male and female rats: prenatally morphine-exposed adult progeny, prenatally saline-exposed adult progeny, and control groups. All male rats were gonadally intact, and female rats were ovariectomized (OVX) in adulthood, and half of them were injected with estradiol benzoate (EB). All animals were exposed to a cold water stressor daily for 2 weeks and tested before (baseline) and after (stress effects) the chronic cold water stressor in a swim test and an open field test. In the swim test, both adult males and OVX, EB-treated adult females born to mothers injected with morphine or saline displayed more floating behavior during the swim test than their controls, both before and after the cold water stressor. Male rats exposed to morphine or saline prenatally also spent more time struggling during the swim tests than controls, and this was further increased after the cold water stressor. In the open field test, males and OVX, EB-treated females born to morphine- or saline-injected mothers were less active and displayed fewer rearings than controls. No differences were observed in OVX females as a result of prenatal injections. Thus, the present study demonstrates that maternal injections, regardless of injection content, induce long-lasting effects on stress responsiveness in adult progeny.

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.

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.

Prenatal morphine exposure alters estrogen regulation of κ 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.