β-endorphin and methionine-enkephalin levels in discrete brain regions, spinal cord, pituitary gland and plasma of morphine tolerant-dependent and abstinent rats

The effect of morphine tolerance-dependence, protracted and naloxone-precipitated abstinence on the levels of β-endorphin and methionine-enkephalin in discrete brain regions, spinal cord, pituitary gland and plasma was determined in the male Sprague-Dawley rats. Among the brain regions examined, the levels of β-endorphin in descending order were: hypothalamus, amygdala, midbrain, hippocampus, corpus striatum, pons and medulla and cortex. The levels of β-endorphin in midbrain, hypothalamus, and pituitary of morphine tolerant-dependent rats were decreased significantly. During protracted withdrawal β-endorphin levels were decreased in amygdala, spinal cord and pituitary. During naloxone-precipitated abstinence β-endorphin levels were increased in corpus striatum, midbrain and cortex. In addition, in naloxone-precipitated abstinence β-endorphin levels were decreased in pituitary gland and hippocampus but increased in plasma. The levels of methionine-enkephalin in brain regions in decreasing order were: corpus striatum, pons and medulla, amygdala, hypothalamus, midbrain, hippocampus and cortex. The levels of methionine-enkephalin in pons and medulla, amygdala, hippocampus and pituitary gland were decreased in morphine tolerant-dependent rats. During protracted abstinence from morphine, methionine-enkephalin levels in spinal cord, amygdala, pons and medulla, midbrain, cortex, corpus striatum and pituitary gland were decreased. The levels of methionine-enkephalin in hypothalamus and corpus striatum of naloxone-precipitated abstinent rats were increased but were decreased in amygdala and pituitary gland. These results suggest that during morphine tolerance-dependence and during protracted abstinence β-endorphin and methionine-enkephalin levels in discrete brain regions and pituitary gland are decreased. During precipitated abstinence β-endorphin levels are increased in brain regions (except hippocampus) and plasma but decreased in pituitary, whereas methionine-enkephalin levels in amygdala and pituitary gland are decreased except in corpus striatum and hypothalamus where they are increased. The pituitary levels of β-endorphin where reduced in all three conditions. However, the levels after withdrawal were not significantly different from those in tolerant—dependent animals.     

Distribution of morphine in brain regions, spinal cord and serum following intravenous injection to morphine tolerant rats

In order to determine the possible contribution of altered distribution of morphine in the morphine tolerance process, the distribution of morphine was studied in brain regions and spinal cord, following its intravenous administration. Male Sprague-Dawley rats were made tolerant to morphine by implanting 6 morphine pellets, each containing 75 mg of morphine base, for 7 days. Seventy-two hours after the removal of the pellets, a time when serum morphine levels were negligible or absent and yet tolerance to the pharmacological effects of morphine was present, morphine (10 mg/kg, i.v.) was injected in placebo and morphine pellet implanted rats. At various times (5, 30, 60, 120 and 360 min) after the injection of morphine, brain regions (hypothalamus, cortex, hippocampus, midbrain, pons and medulla, striatum and amygdala), spinal cord and serum were collected. The level of morphine in the tissues was determined by using a highly sensitive and specific radioimmunoassay (RIA) method. Five minutes after morphine injection, the concentration of morphine was the highest in the hypothalamus and the lowest in amygdala. The concentration of morphine in hypothalamus, pons and medulla, hippocampus and midbrain of morphine tolerant rats was smaller than in placebo pellet implanted rats. The tissue to serum ratio of morphine in the hypothalamus, hippocampus, striatum, midbrain and cortex were also smaller in morphine tolerant than in non-tolerant rats. The concentration of morphine in brain regions with time did not exhibit linearity. At other time intervals like 30 and 60 min, the concentration of morphine in several brain regions and spinal cord was significantly higher in morphine tolerant than in non-tolerant rats.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effect of morphine tolerance dependence and abstinence on immunoreactive dynorphin (1–13) levels in discrete brain regions, spinal cord, pituitary gland and peripheral tissues of the rat

The effect of morphine tolerance dependence and protracted abstinence on the levels of dynorphin (1–13) in discrete brain regions, spinal cord, pituitary gland and peripheral tissues was determined in male Sprague-Dawley rats. Of all the tissues examined, the highest level of dynorphin (1–13) was found to be in the pituitary gland. Among the brain regions and spinal cord examined, the levels of dynorphin (1–13) in descending order were: hypothalamus, spinal cord, midbrain, pons and medulla, hippocampus, cortex, amygdala and striatum. The descending order for the levels of dynorphin (1–13) in peripheral tissues was: adrenals, heart and kidneys. In morphine tolerant rats, the levels of dynorphin (1–13) increased in amygdala but were decreased in pons and medulla. In morphine abstinent rats, the levels of dynorphin (1–13) were increased in amygdala, hypothalamus and hippocampus. The levels of dynorphin (1–13) were increased in pituitary but decreased in spinal cord and remained so even during protracted abstinence. The levels of dynorphin (1–13) in the peripheral tissues of morphine tolerant rats were unaffected. However, in the heart and kidneys of morphine abstinent rats, the levels of dynorphin (1–13) were increased significantly. It is concluded that both morphine tolerance and abstinence modify the levels of dynorphin (1–13) in pituitary, central and peripheral tissues. Morphine abstinence differed from non-abstinence process in that there were additional changes (increases) in the levels of dynorphin (1–13) in brain regions (hypothalamus and hippocampus) and peripheral tissues (heart and kidneys) and may contribute to the symptoms of the morphine abstinence syndrome. The lower levels of dynorphin (1–13) in spinal cord may be responsible for the potentiation of morphine effects by κ-opiate agonist in morphine tolerant dependent rodents.     

Opioid peptides in pituitary gland, brain regions and peripheral tissues of spontaneously hypertensive and Wistar-Kyoto normotensive rats

The concentrations of β-endorphin (β-END), dynorphin (DYN) and methionine-enkephalin (MEK) in pituitary, brain regions, heart, kidney and adrenal of 8 week old male spontaneously hypertensive (SHR) and Wistar-Kyoto (WKY) normotensive rats were determined by radioimmunoassay and compared. The brain regions examined were hypothalamus, striatum, pons + medulla, midbrain and cortex. The concentration of β-END in pituitary of SHR rats was 49% higher than those of WKY rats. The concentration of β-END in the striatum of SHR rats was 71% lower as compared to WKY rats. The concentration of β-END in the heart, adrenals and kidney of SHR rats was significantly lower (92. 48 and 57%, respectively), than those of WKY rat tissues. The concentration of DYN in pituitary, striatum and heart were lower by 38, 55 and 46%, respectively, in SHR compared to WKY rats, but in hypothalamus it was greater (33%) than in WKY rats. The concentration of DYN in other brain areas and in kidney and adrenal did not differ. The tissues of SHR and WKY rats which showed significant difference in the concentration of MEK were pituitary, pons + medulla, cerebral cortex and adrenals. The concentration of MEK was greater in SHR rats with pons + medulla, cortex and adrenals showing 33, 40, 268% higher levels, respectively, over the WKY rat tissues. However, the concentration of MEK in pituitary of SHR rats was 40% lower than that of WKY rats. These studies suggest that the endogenous opioid peptides of both central and peripheral tissues may be important in the regulation of blood pressure in SHR rats.     

Effects of estrogen and progesterone on plasma gonadotropins and on catecholamine levels and turnover in discrete brain regions of ovariectomized rats.

Estradiol benzoate (EB) was administered, either alone or followed 48 h later by progesterone to ovariectomized rats. Plasma gonadotropins (FSH and LH) and steady state levels of norepinephrine (NE) and dopamine (DA) in 17 individual brain nuclei were assayed. In addition, catecholamines were measured after administration of the synthesis inhibitor alpha-methyltyrosine (alpha-MT) in order to assess hormonal influences on turnover. Treatment with EB, which lowered plasma FSH and LH, reduced the depletion of NE produced by alpha-MT in the lateral septum, interstitial nucleus of the stria terminalis, and central gray catecholamine area, and reduced the depletion of DA in the nucleus of the tractus diagonalis. EB enhanced NE depletion in the periventricular and anterior hypothalamic nuclei, and raised steady state levels of NE in the medial amygdaloid nucleus. These effects were reversed by subsequent treatment with progesterone, which stimulated FSH and LH release. EB plus progesterone enhanced the alpha-MT-induced depletion of NE over that observed with EB alone in the arcuate nucleus, and similarly enhanced DA depletion in the interstitial nucleus of the stria terminalis. EB plus progesterone prevented the depletion of NE by alpha-MT in the paraventricular and ventromedial nuclei, and also lowered resting NE levels in the paraventricular nucleus. The results suggest that catecholamine neurons in several discrete brain regions participate in the stimulatory and inhibitory feedback effects of ovarian hormones on gonadotropin secretion, and perhaps also on the hormonal induction of sexual receptivity.     

Effects of mediobasal hypothalamic lesion on immunoreactive ACTH/beta-endorphin levels in cerebrospinal fluid, in discrete brain regions, in plasma, and in pituitary of the rat.

One week after complete destruction of the mediobasal hypothalamus, immunoreactive adrenocorticotropin (ACTH) and beta-endorphin levels were determined in cerebrospinal fluid, trunk blood, as well as in brain and pituitary tissue samples collected from anaesthetized and cisternally cannulated rats. Control rats were sham operated. In lesioned rats we observed: (a) 60% decrease in the immunoreactive beta-endorphin concentrations in the cerebrospinal fluid, (b) decreased immunoreactive ACTH and beta-endorphin levels in the hypothalamus, in the thalamus and in the amygdala, (c) unaffected immunoreactive ACTH/beta-endorphin levels in the septum and in the hippocampus, (d) decreased immunoreactive beta-endorphin levels both in the anterior and neurointermediate pituitary but unchanged immunoreactive ACTH contents in the anterior lobe, and (e) unaffected immunoreactive ACTH and beta-endorphin levels in the plasma under stressful conditions. From these findings the following conclusions can be drawn: (1) more than 50% of the beta-endorphin-like peptide content of the cerebrospinal fluid originates from the periventricular nuclei of the hypothalamus and thalamus in the rat; (2) the loss of the hypothalamic control probably enhances the intracellular proteolytic degradation of beta-endorphin both in the anterior and neurointermediate pituitary lobe; (3) rats with mediobasal hypothalamic lesion cannot react to the stressful stimuli of ether anaesthesia or cisternal cannulation with elevated plasma immunoreactive ACTH and beta-endorphin levels.     

The effect of U-50,488H tolerance-dependence and abstinence on the levels of dynorphin (1–13) in brain regions, spinal cord, pituitary gland and peripheral tissues of the rat

Male Sprague-Dawley rats were rendered tolerant to and physically dependent on U-50,488H, a κ-opiate agonist, by injecting 25 mg/kg of the drug intraperitoneally twice a day for 4 days. Two sets of rats were used. Rats labeled as tolerant-dependent were injected with U-50,488H (25 mg/kg) 1 h before sacrificing on day 5, whereas the abstinent rats were sacrificed on day 5 without the injection of U-50,488H. Of all the tissues examined, the pituitary gland had the highest level of dynorphin (1–13), whereas the heart had the lowest level. The levels of dynorphin (1–13) increased in the hypothalamus, hippocampus and pons/medulla of U-50,488H tolerant-dependent rats, whereas in abstinent rats the levels of dynorphin (1–13) were elevated only in the midbrain. The levels of dynorphin (1–13) in the pituitary gland of U-50,488H tolerant-dependent or abstinent rats were unchanged. In peripheral tissues, the levels of dynorphin (1–13) in the heart of U-50,488H tolerant-dependent rats were increased. In the abstinent rats they were elevated in the adrenals, spleen, and the heart but were decreased in the kidneys. Compared to morphine tolerant-dependent and abstinent rats, significant differences in the levels of dynorphin (1–13) in tissues of 50,488H tolerant-dependent and abstinent rats were observed and may explain many pharmacological differences in the μ- and κ-opiate induced tolerance-dependence and abstinence processes.     

Influence of halothane and methoxyflurane on regional brain and spinal cord concentrations of methionine-enkephalin in the rat

Rats were exposed to either oxygen (controls), 1.5% halothane in oxygen, or methoxyflurane (0.5%) in oxygen over a period of 2 h, then sacrificed at the end of exposure (2-h group), 4 h after removal from environmental chamber (4-h group), or at 24 h following anesthetic exposure (24-h group). Pituitary (excluding the neural lobe, Pit), brain, and spinal cord areas were isolated and processed with Met-enkephalin tissue concentrations determined. In halothane-exposed animals, Met-enkephalin concentrations in pit and across CNS areas studied were significantly lower at 2 h following anesthetic exposure than in control animals. Concentrations of Met-enkephalin in many areas of CNS and Pit of 4-h group approached control levels. Concentrations of Met-enkephalin in all areas studied except spinal cord returned to basal levels by 24 h following halothane exposure. Exposure to methoxyflurane resulted in less dramatic changes in Met-enkephalin concentrations across CNS regions examined. Exposure to methoxyflurane resulted in significant decreases in Met-enkephalin levels in olfactory bulb, thalamus, and hippocampus only. Met-Enkephalin levels did not change significantly in other areas of the central nervous system following methoxyflurane exposure. These results indicate that halothane and methoxyflurane may have differential effects on the endogenous opioid system.     

吗啡精神依赖大鼠相关脑区细胞外氨基酸类神经递质的变化

目的 研究吗啡精神依赖大鼠伏隔核(NAc)、苍白球腹侧核(VP)以及中脑腹侧被盖区(VTA)中细胞外谷氨酸(Glu)和γ-氨基丁酸(GABA)含量的变化. 方法 18只Wisar大鼠按照随机数字表法分为对照组(8只)和吗啡组(10只).吗啡组大鼠每日皮下注射盐酸吗啡,并进行位置偏爱(CPP)训练,10 d后停药,戒断躯体依赖后行CPP评分;对照组大鼠在相同时间给予同体积的生理盐水皮下注射10d.停药一周后用微透析探针对大鼠NAc、VP和VTA行微透析,用高效液相色谱法(HPLC)测定透析液中的Glu和GABA的含量. 结果 吗啡组NAc中细胞外Glu含量明显高于对照组,差异有统计学意义(P＜0.05),两组VP和VTA中细胞外Glu和GABA含量比较差异有统计学意义. 结论 Glu在维持精神依赖的过程中发挥重要作用.     

The ribosomal synthesis and release of methionine-enkephalin and substance P by spinal cord and brain cell cultures

Peptide synthesis and release in primary dissociated nerve cell cultures have been studied using a very high specific activity, radiolabeled amino acid precursor, high-performance liquid chromatography and amino acid sequencing of purified peptides. The combined use of these methods has resulted in the rigorous demonstration of ribosomal synthesis of methionine-enkephalin by spinal cord cells, the basal release of methionine-enkephalin by spinal cord and substance P by brain cells, the tetrodotoxin sensitivity of this spontaneous release, and the potassium depolarization induced release of both peptides in a calcium dependent manner. This approach to the study of the development and biological chemistry of peptidergic neurons is recommended as a model because of the accessibility of the cells under conditions of ongoing physiologic function and the unequivocal nature of the chemical characterization of synthesized peptides.     

Tyrosine hydroxylase activity in discrete brain regions of depression model rats

Tyrosine hydroxylase (TH) activity was measured in discrete brain regions of rats during short-term forced running stress (FRS). TH activity was also determined in a depression-like state and in a recovered state after a long-term FRS. Under the short-term FRS, the TH activity showed a significant increase in the locus ceruleus, certain limbic regions and tuberoinfundibular system. In the depression-like state, however, there was a significant decrease in the locus ceruleus and certain limbic regions, but a significant increase was seen in the median eminence. The TH activity in recovered rats showed no difference from the level in the controls. These findings demonstrate an adaptive increase in the TH activity in relation to stress, and may also indicate a failure of adaptation in the depression-like state.     

Differential effects of morphine on histamine metabolism in brain and spinal cord of mice

The effects of morphine on the levels of histamine (HA), its metabolite tele-methylhistamine (t-MH) and on t-MH synthesis rates (thought to be indicative of neuronal HA release) were investigated in brain regions and spinal cords of DBA/2J (DBA) and C57/BL6 (C57) mice, two strains known to differ in their sensitivity to morphine. In DBA (a strain highly sensitive to morphine antinociception), morphine (10 mg/kg, s.c.) had no effect on brain regional t-MH or HA levels, but produced a generalized inhibition of regional t-MH synthesis rates ranging from 11 to 53%. The monoamine oxidase (MAO) inhibitor pargyline (used to estimate t-MH synthesis rates) had no effect on HA or t-MH levels in the DBA or C57 spinal cord, indicating the absence of detectable spinal HA turnover. Morphine (10 mg/kg) had no effect on DBA or C57 spinal cord HA or t-MH levels, but significantly increased t-MH synthesis in the DBA but not in the C57 spinal cord. These results suggest that in DBA mice, antinociceptive doses of morphine inhibit HA release in brain, and promote the release of HA from spinal cord. Neither effect was found in C57 mice, which are resistant to morphine antinociception. The relevance of these actions to previous studies showing the blockade of opiate antinociception by H2 antagonists remains to be established.     

Changes in brain and spinal tryptophan and 5-hydroxy-indoleacetic acid levels follwing acute morphine administration in normal and arthritic rats

The effects of morphine (10 mg/kg/s.c.) on tryptophan (TRP), 5-hydroxy-tryptamine (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) levels were studied in normal and arthritic rats.

(1) In normal rats morphine induced a discrete but significant increase of 5-HIAA levels in the forebrain and the spinal cord.

(2) By contrast, in rats suffering from experimentally induced arthritis large modifications were apparent. The basal levels of TRP, 5-HT and 5-HIAA were significantly higher than in normal rats. Morphine induced clear increases of 5-HIAA and TRP in the forebrain, the brain stem and the spinal cord, without any modification of 5-HT. The effects were dose-dependent and suppressed by naloxone (1 mg/kg/ i.m.). Statistical analysis clearly revealed that arthritic rats were much more sensitive to morphine.

The results support the hypothesis of an activation of a 5-HT descending pathway by morphine which parallels the activationn of theascending pathway previously demonstrated by several authors and confirmed here.     

Recovery of spinal cord volume in postnatal rats following prenatal exposure to morphine

A previous study has shown that the spinal volume of 18-day fetal rats is decreased by 20% after maternal administration of morphine on gestation days 12-18. In the present study, the volume of the first thoracic spinal cord segment was measured in offspring of morphine or saline-injected and pairfed dams on days 6, 15 and 80 postnatally. The following volumes were measured within the segment: hemisegment, gray matter, white matter, dorsal horn, ventral horn and length. On the sixth day postnatally, the hemisegment, gray matter and dorsal horns are significantly reduced in morphine-treated and pairfed offspring. By the fifteenth day postnatally, only the gray/white ratio is reduced while the length of the segment is significantly increased in morphine-treated fetuses. The spinal measurements in pairfed offspring are normal by 15 days postnatally. These results indicate that the effect of morphine on developing spinal cord may be partially due to undernutrition; however, morphine causes a more pronounced and longer-lasting effect than undernutrition alone.     

Developmental changes in serotonin levels in the chick spinal cord and brain

Developmental changes in 5-hydroxytryptamine (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) in the developing chick spinal cord and brain were examined using high-performance liquid chromatography with electrochemical detection and immunohistochemistry. On embryonic day (E)6 only small amounts of 5-HT (0.086 ng) and 5-HIAA (0.0144 ng) were found in the spinal cord. By contrast, large amounts of 5-HT (x30) and 5-HIAA (x60) were detected in non-neuronal tissue outside the spinal cord; a similar distribution of 5-HT was also detected by immunohistochemistry. Up to E10, the highest concentrations of 5-HT in the spinal cord were found in the cervical region, followed by the thoracic and lumbar regions. In embryos older than E16, as well as in posthatched chicks, however, the highest and lowest concentrations of 5-HT were found in the lumbar and thoracic spinal cord, respectively. The concentration of spinal cord 5-HT reached maximal values on posthatching day (P)7, after which there was a marked decrease. By P120, 5-HT levels in the spinal cord decreased to the same level as on E10-E16. Concentrations in the brain, however, gradually increased with development. The basic pattern of development of 5-HIAA was similar to that of 5-HT.     

Gonadal influences on spinal cord and brain monoamines in male rats

Concentrations of norepinephrine (NE), dopamine (DA), 3,4-dihydroxy phenylacetic acid (DOPAC), serotonin (HT) and 5-hydroxyindoleacetic acid (HIAA) were measured by high-performance liquid chromatography-electrical detection (HPLC-ED) in homogenates of lumbosacral spinal cord, mediobasal hypothalamus and cerebral cortex of male rats. The effects of castration and testosterone propionate (TP) (20 micrograms/day X 2 days) were compared. Castrated animals had the highest levels of DA and DOPAC in the cerebral cortex and NE, HT and HIAA in the spinal cord, as well as decreased hypothalamic DOPAC. Testosterone treatment returned spinal cord monoamine concentrations to intact control levels. These findings point to the spinopetal monoaminergic pathways as sensitive targets for androgen action.