An iontophoretic survey of opioid peptide actions in the rat limbic system: in search of opiate epileptogenic mechanisms

Iontophoretic and micropressure drug application and lesion techniques were used to investigate the cellular source of rat limbic system epileptiform responses to opioid peptides [19]. Iontophoretically applied morphine, methionine enkephalin or beta-endorphin inhibited the spontaneous or glutamate-activated firing of the great majority of single neurons in medial and lateral septum, amygdala and cingulate cortex. These inhibitions in firing were antagonized by iontophoresis of naloxone. In contrast to inhibitory effects in other limbic areas, morphine and the opioid peptides predominantly excited CA1 and CA3 pyramidal neurons in a naloxone-sensitive manner, as previously reported [36]. On rare occasions, iontophoretically applied beta-endorphin evoked repetitive waveforms similar to interictal population EPSPs or spikes. Micropressure application of opiates and peptides also excited hippocampal neurons indicating such responses were not current-induced artefacts. The possible role of the excitatory cholinergic septal hippocampal pathway in the facilitatory response of hippocampal units to the opiates was tested with iontophoretically applied atropine and scopolamine, or lesions of septal nuclei. None of these manipulations reduced the opioid-induced excitations; rather, septal lesions enhanced excitatory and epileptiform responses to the opiates. These results support the hypothesis that opiate-evoked epileptiform activity in the limbic system arises from enhanced pyramidal cell activity in the hippocampal formation, probably by a non-cholinergic mechanism.     

Naloxone and the kindling of seizures.

In view of evidence indicating that endogenous opiate-like peptides have epileptiform effects, we examined the effect of the opiate antagonist naloxone on the kindling of seizures produced by repeated electrical stimulation of the amygdala or the caudate nucleus in rats. Naloxone had no effect on the threshold for local after-discharge in the two areas and failed to retard the rate of kindling of clinical seizures. These results suggest that an interaction of opiate-like peptides with central opiate receptors does not play any critical role in the kindling of seizures.     

Opioid pharmacology in the rat hippocampal slice

The ability of several opioids in potentiating the synaptic activation of CA1 pyramidal cells in the rat hippocampal slice were compared. Morphine and the opioid peptides, (D-ala2, D-leu5)-enkephalin (DADL), morphiceptin, beta-endorphin, and Tyr-D-Ser-Gly-Phe-Leu-Thr (DSThr) caused a concentration-dependent, naloxone-reversible shift to the left in the input-output (IO) curve constructed by plotting the population spike as a function of the field EPSP. These opioids then produced an increase in the size of the population spike while leaving the EPSP unaffected. In contrast, the kappa agonist prototype, ethylketazocine, had no effect on the IO curve when perfused in concentrations up to 10 microM. The rank order of potency for the opioids in the CA1 region of the hippocampus was DADL greater than DSThr greater than beta-endorphin greater than morphiceptin greater than morphine much greater than ethylketazocine. Thus, opioids that are more specific for delta opiate receptors were the most potent and mu receptor agonists, the least potent in this action. Taken together with previous studies suggesting that morphine and DADL may interact with a common opiate receptor in the CA1 region, the results are consistent with the notion that these epileptiform effects may be primarily mediated by delta opiate receptors in this area although the potency of morphiceptin indicates that mu receptors play some role in this effect.     

Interaction of various types of opiate receptors of cortical neurons in the turtle during activation by specific agonists

The influence of polycomponent solutions of agonists of opiate mu-, delta, chi- and sigma-receptors (morphine, D-Ala2, D- Ley5 -enkephalin, bremazocine, SKF 10,047) and of met-enkephalin on the habituation of orthodromic evoked potential in the visual cortical area was studied in turtles. Interaction between the different types of opiate receptors was observed at their combined activation. The interaction resulted in an enhancement or attenuation of modulation of separate phases of evoked potential habituation which differed from simple sum of effects during isolated activation of each type of receptors.     

Effects of morphine and methionine-enkephalin on the smooth muscle tonus and the contraction induced by transmural stimulation in the carp (Cyprinus carpio) intestinal bulb

The effects of morphine and methionine-enkephalin (met-enkephalin) on the smooth muscle tonus and the contraction induced by transmural stimulation were investigated in the isolated intestinal bulb of carp in vitro. Morphine (30 nM-3 microM) and met-enkephalin (3 nM-5 microM) caused dose-dependent non-sustained contraction. Naloxone (10 nM) inhibited the contraction induced by morphine or met-enkephalin in a competitive manner. Tetrodotoxin (400 nM) or atropine (500 nM) did not inhibit the contraction induced by morphine or met-enkephalin. Cooling of the bath fluid from 20 to 10 degrees C decreased nicotine- and transmural stimulation-induced contraction. But met-enkephalin-induced contraction was not affected. Transmural stimulation-induced contraction (3 Hz) was not affected by pretreatment with morphine, met-enkephalin or naloxone. The results demonstrated that morphine or met-enkephalin caused contraction of the smooth muscle directly through the activation of opiate receptors on the smooth muscle cells and neither morphine nor met-enkephalin regulated the cholinergic neurotransmission presynaptically.     

Modulation of opioid system in C57 mice after repeated treatment with morphine and naloxone: biochemical and behavioral correlates

C57 BL/6J (C57) mice display a particular pattern of responses following morphine administration, such as a rapid development of tolerance to the pharmacological action of the opiate and an increase in locomotor activity after a single injection of the drug. We have measured met-enkephalin content and the responsiveness of different opiate receptors after repeated administration of morphine and naloxone. Prolonged morphine administration changes neither met-enkephalin levels, nor the density of the opiate receptors in mice brain. In contrast repeated administration of the opiate antagonist naloxone, produced a marked increase in the number of 3H- DHM and 3H- DADLE binding sites in striatum and brainstem without modifying met-enkephalin concentrations. Behavioral studies have indicated that the morphine-induced increase in locomotor activity is enhanced in naloxone pretreated mice, thus suggesting a possible correlation between the behavioral response to morphine in C57 mice and the higher number of opiate receptors in the striatum.     

Effect of met-enkephalin and morphine on gastric secretion and blood flow

The effects of met-enkephalin and morphine on gastric acid and pepsin secretion and gastric mucosal and total blood flow were studied in anaesthetized dogs with an in vivo chambered secretion stomach preparation. It was found that both agents infused intraarterially caused an increase in histamine-induced acid and pepsin secretion and mucosal and total blood flow. The above responses were significantly blocked by naloxone and nalorphine. In the resting stomach both opiates did not induce secretory changes but they increased mucosal and total blood flow. Met-enkephalin and morphine were also effective after intravenous administration. Met-enkephalin but not morphine fails to stimulate acid secretion if given into the portal vein. The likely mechanism of action of opiates on gastric secretion is discussed and a hypothesis of existence of opiate receptors in the gastric wall is presented.     

Modulation of spontaneous seizures in the mongolian gerbil: effects of beta-endorphin

Intraventricular injection of beta-endorphin (0.1-3 micrograms) into gerbils from the UCLA seizure sensitive strain reduced the incidence and severity of spontaneous epileptiform seizures, both the motor manifestations and the preceding high voltage focal spiking and accompanying seizure activity in the cortical EEG. This "'anticonvulsant" effect of beta-endorphin was prevented by prior administration of naloxone (1 mg . kg-1 IP). These findings suggest that the endogenous opioid peptide may be involved in the normal suppression of the epileptic diathesis in these animals during the interictal periods.     

Antagonism of diazepam-induced feeding in rats by antisera to opioid peptides

Diazepam-induced feeding in rats is antagonized not only by the opiate antagonist naloxone but also intraventricular administration of specific antisera to the endogenous opioid peptides met-enkephalin or beta-endorphin. Pituitary beta-endorphin is probably not implicated in the diazepam effect since blockade with the glucocorticoid dexamethasone of the release of beta-endorphin from the anterior pituitary does not modify the diazepam-induced feeding, which is however prevented by TRH, a suggested physiological antagonist of some of the effects of opioid peptides. The possible central participation of both beta-endorphin and met-enkephalin in the ingestive behavior induced by diazepam gives further support to the postulated physiological role of endogenous opioids in appetite regulation.     

家兔伏核—杏仁核神经通路在吗啡镇痛中的作用

用辐射热照射家兔鼻嘴侧部皮肤,测量其躲避反应潜伏期作为痛反应阈,简称痛阈。通过预先埋植的慢性套管向伏核或杏仁核内进行注射,结果表明:(1)在家兔的伏核内微量注射吗啡可产生镇痛作用,该作用可被杏仁核内注射纳洛酮所削弱,并有量效依从关系;在杏仁核内注射甲啡肽抗血清(ME AS)或β-內啡肽抗血清(β-EP AS)亦可削弱上述镇痛作用;(2)在杏仁核内微量注射吗啡可产生镇痛作用,此作用不能被伏核内注射纳洛酮所阻断;(3)在伏核内注射吗啡所产生的镇痛作用可被同一部位注射γ-氨基丁酸(GAEA)受体阻断剂氯甲基荷包牡丹碱所增强,被 GABA 受体激动剂异鹅羔胺所削弱。上述结果提示:在家兔脑内从伏核到杏仁核可能存在一条与镇痛有关的神经通路,伏核内的阿片样物质及杏仁核内的甲啡肽,β-内啡肽可能参与镇痛信息的传递,而伏核内的 GABA 可能有对抗吗啡镇痛的作用。     

Effects of morphine, enkephalins and naloxone on postprandial gastric acid secretion, gastric emptying and gastrin release in dogs

The effects of intravenous infusions of morphine, met-enkephalin and leu-enkephalin on gastric acid secretion, gastrin release and gastric emptying were investigated in four dogs with gastric cannulas stimulated by a liquid peptone meal. The actions of a potent opiate antagonist, naloxone, used alone or combined with opiates were also studied. Morphine, met-and leu-enkephalin decreased the fractional gastric emptying rate. Acid secretion was decreased by enkephalins and increased by high doses of morphine. Enkephalins and to a lesser degree morphine inhibited gastrin release during the first hour following the administration of the meal. Only leu-enkephalin decreases significantly the integrated gastrin response. Naloxone at the doses used antagonized partly or totally the effects of opiates on gastric emptying but not those on gastric secretion or gastrin release. Naloxone infused alone had no significant effect on the gastric functions tested. These studies indicate that in dogs stimulated by a liquid test meal, enkephalins inhibit gastric emptying, acid secretion and gastrin release. Morphine inhibits gastric emptying and gastrin release and enhances acid secretion.     

Evidence for intrinsic regulation of met-enkephalin-immunoreactivity in gastroenteropancreatic tissues of the rat

In a study whether gastrointestinal endogenous opioids can be modified by vagal denervation or by pharmacological application of an opiate, we examined met-enkephalin-immunoreactivity in gastrointestinal tissue in rats with and without truncal vagotomy and with and without subcutaneously implanted morphine pellets. The immunoreactivity of the tissue extracts gave dose-response lines in the radioimmunoassay for met-enkephalin which were near parallel to that for the standard. On Sephadex chromatography the met-enkephalin immunoreactivity eluted at a position similar to synthetic met-enkephalin. Tissue concentration of met-enkephalin immunoreactivity was not significantly different from the respective control after vagotomy and after morphine treatment. Total gastric met-enkephalin immunoreactive content increased significantly after vagotomy in line with gastric hypertrophy occurring after vagotomy without a drainage procedure. From these results it is concluded that met-enkephalin immunoreactivity in the rat gastrointestinal tract is regulated intrinsically, it is neither altered by vagal denervation nor by exogenous opiate administration.     

Urethane anesthesia blocks the development and expression of kindled seizures

The effect of anesthetic and subanesthetic doses of urethane on the development of amygdala kindled seizures and on the expression of previously kindled seizures was studied in hooded rats. An anesthetic dose of urethane (1.5 g/kg) almost completely eliminated evoked afterdischarge and completely eliminated convulsive behavior in both groups. It also eliminated the seizure response to pentylenetetrazol. Subanesthetic doses of urethane (0.25 and 0.5 g/kg) strongly attenuated the expression of previously kindled seizures. These results suggest that urethane may not be an appropriate anesthetic for the study of epileptiform phenomena.     

Potencies of normorphine and met-enkephalin in the vas deferens of different strains of mice.

The concentration of normorphine causing a 50 per cent inhibition (IC₅₀) of electrically induced twitches in the vas deferens from seven strains of mice varied over a 13-fold range, BALB/cKB being the most, C57BL/6J the least sensitive. There was no significant correlation between the IC₅₀'s of normorphine and met-enkephalin. In the sensitive BALB/cKB mice, both normorphine and met-enkephalin were more effective inhibiting contractions evoked by 0.1 Hz than by 0.01 or 1.2 Hz stimulation. This difference was not observed in the insensitive C57BL/6J mice. Naloxone was purely an antagonist against both normorphine and met-enkephalin in BALB/cKB mice but in low concentration it potentiated the inhibitory effect of both normorphine and met-enkephalin in C57BL/6J mice. Results suggest that qualitative differences in opiate receptors and differences in transmitter release mechanism contribute to the variable sensitivity to morphine of the vas deferens from different strains of mice.     

Antinociceptive effects of intrathecally administered human beta-endorphin in the rat and cat

Rats chronically implanted with intrathecal catheters displayed a dose-dependent increase in the hot-plate and tail-flick response latencies following the injection of human beta-endorphin into the lumbar spinal subarachnoid space through the indwelling catheter. beta-Endorphin was approximately 25 times more potent than morphine on a molar basis. Matching morphine and beta-endorphin doses such that approximately equal submaximal submaximal effects occurred, it was observed that the antinociception produced by beta-endorphin lasted approximately three times longer than that produced by morphine. Experiments with intrathecal injection of beta-endorphin into the spinal subarachnoid space of cats fitted with intrathecal catheters also revealed a potent antinociceptive effect which was completely antagonized by naloxone. In the rats, naloxone administered systemically in doses of 10--100 microgram/kg produced a parallel shift in the dose-response curves of both nociceptive measures suggesting a competitive antagonism. Using a dose ratio analysis, an in vivo pA2 of 7.1 for naloxone was obtained. These data and those derived from previous work based on the pA2 suggest that the interaction of morphine, certain pentapeptides, and beta-endorphin is the same with regard to the spinal opiate receptor population mediating behaviorally defined analgesia.     

Opioid activity of delta O-endorphin in the guinea pig ileum.

The oligopeptides beta- and delta O-endorphin were isolated from porcine and bovine pituitary respectively. Their opiate activity was determined in the guinea pig ileum and compared to that of the pentapeptide methionine-enkephalin and morphine. The rank order of opioid activity was found to be: morphine greater than beta-endorphin = Met-enkephalin greater than delta O-Endorphin which lacks the four C-terminal amino acids of beta-endorphin displayed 60% of the activity of beta-endorphin. These results indicate, that C-terminal amino acids contribute little to the affinity of beta-endorphin for opiate receptors in the guinea pig ileum.     

Melanin-concentrating hormone (MCH) modifies memory retention in rats

The purpose of the present study was to evaluate the possible effect of melanin-concentrating hormone (MCH) on learning and memory by using the one-trial step-down inhibitory avoidance test in rats. The peptide was infused into hippocampus, amygdala, and entorhinal cortex. MCH caused retrograde facilitation when given at 0 or 4 h post-training into hippocampus, but only at 0 h into amygdala. From these results, it seems that MCH modulates memory early after training by acting on both the amygdala and hippocampus and, 4 h after training, on the hippocampus.     

Melanin-concentrating hormone (MCH) modifies memory retention in rats

The purpose of the present study was to evaluate the possible effect of melanin-concentrating hormone (MCH) on learning and memory by using the one-trial step-down inhibitory avoidance test in rats. The peptide was infused into hippocampus, amygdala, and entorhinal cortex. MCH caused retrograde facilitation when given at 0 or 4 h post-training into hippocampus, but only at 0 h into amygdala. From these results, it seems that MCH modulates memory early after training by acting on both the amygdala and hippocampus and, 4 h after training, on the hippocampus.     

The role of the opiate mechanisms of the hippocampus and substantia nigra in the behavioral and convulsive disorders in picrotoxin-induced kindling]

It was shown in the experiments on rats that the repeated picrotoxin administration resulted in the kindling of generalized seizures. Generalized convulsions were followed by the development of either postictal depression or explosiveness. The injection of mu-opiate agonist met-enkephalin into hippocampus of kindled rats resulted in the increase in the severity of seizure reactions which were induced by picrotoxin and also in the increase in the number of animals with postictal explosiveness. The injection of dynorphin-A-1-13 (kappa-opiate agonist) into substantia nigra reticulata induced the locomotor depression which was like one in postictal period and resulted in the decrease of picrotoxin-induced seizures severity. It was concluded that mu-opiate system of hippocampus took part in the formation of generator of pathologically enhanced excitation in the structure during kindling and the development of seizure syndrome, providing also the postictal explosiveness. Kappa-opiate system of substantia nigra plays an important role in the activation of the antiepileptic system, limitation of seizures and the development of postictal depression.     

Involvement of opioidergic system of the ventral hippocampus, the nucleus accumbens or the central amygdala in anxiety-related behavior

In the present study, the influence of opioidergic system of the ventral hippocampus, the nucleus accumbens or the central amygdala on anxiety-related behaviour was investigated in rats. As a model of anxiety, the elevated plus maze which is a useful test to investigate the effects of anxiogenic or anxiolytic drugs in rodents was used. Bilateral microinjection of different doses of morphine (2.5, 5 and 7.5 microg/rat) into the ventral hippocampus or the nucleus accumbens increased the percentage of open arm time (%OAT) and open arm entries (%OAE) but not locomotor activity, indicating an anxiolytic response. However, intra-central amygdala administration of the opioid did not show any response. On the other hand, microinjection of a dose of naloxone into the ventral hippocampus (2 microg/rat) or the nucleus accumbens (1 microg/rat) increased open arm time (%OAT), but not open arm entry (%OAE) which may indicate an anxiolytic effect. Pre-treatment administration of naloxone (0.5, 1 and 2 microg/rat) reversed the anxiolytic effect of morphine (7.5 microg/rat) injected into the ventral hippocampus in a dose-dependent manner. A dose of the antagonist (1 microg/rat) also reduced the morphine response (2.5 microg/rat) when injected in the nucleus accumbens. In conclusion, it seems that the opioidergic system in the ventral hippocampus and the nucleus accumbens are involved in anxiety-related behaviors and the ventral hippocampus may be the main site of action of the anxiolytic properties of morphine.