Opioids in the perifornical lateral hypothalamus suppress ethanol drinking.

The opioid system is known to enhance motivated behaviors, including ethanol drinking and food ingestion, by acting in various reward-related brain regions, such as the nucleus accumbens, ventral tegmental area and medial hypothalamus. There is indirect evidence, however, suggesting that opioid peptides may act differently in the perifornical lateral hypothalamus (PF/LH), causing a suppression of consummatory behavior. Using brain-cannulated Sprague-Dawley rats trained to voluntarily drink 7% ethanol, the present study tested the hypothesis that opioids in the PF/LH can reduce the consumption of ethanol, with animals receiving PF/LH injections of the δ-opioid receptor agonist D-Ala2-met-enkephalinamide (DALA), the µ-receptor agonist [D-Ala2, N-MePhe4, Gly-ol]-enkephalin (DAMGO), the κ-receptor agonist (±)-trans-U-50,488 methanesulfonate (U-50,488H), or the general opioid antagonist methylated naloxone (m-naloxone). The consumption of ethanol, lab chow, and water was monitored for 4 h after injection. The results showed that the three opioid receptor agonists injected into the PF/LH specifically and significantly reduced ethanol intake, while causing little change in chow or water intake, and the opposite effect, enhanced ethanol intake, was observed with the opioid antagonist. Of the three opioid agonists, the δ-agonist appears to produce the most consistent and long-lasting suppression of consumption. This effect was not observed with injections 2 mm dorsal to this area, focusing attention on the PF/LH as the main site of action. These results suggest that the opioid peptides have a specific role in the PF/LH of reducing ethanol drinking, which is distinct from their more commonly observed appetitive actions in other brain areas. The additional finding, that m-naloxone in the PF/LH stimulates ethanol intake in contrast to its generally suppressive effect in other regions, focuses attention on this hypothalamic area and its distinctive role in contributing to the variable effects sometimes observed with opioid antagonist therapy for alcoholism.

Effect of estradiol on expression and activation of Akt protein in rat hypothalamus exposed to chronic [D-Met2, Pro5]-enkephalinamide treatment.

Adult ovariectomized rats were implanted with [D-Met2, Pro5]-enkephalinamide (ENK)-containing osmotic minipumps. Two hours prior to sacrifice, some animals were treated with estradiol-17beta (E2) at a dose 10 microg/100 g bodyweight (BW). Expression and activation of Akt proteins, nuclear [3H]estradiol binding, and the expression of estrogen receptor alpha (ERalpha) and beta (ERbeta) and of progesterone receptor (PR) were investigated. Estradiol increased the level of activated Akt protein (pAkt473) in the hypothalamus by 52 +/- 11% in comparison to the vehicle-treated controls. No such effect of E2 was observed 24 and 48 h after ENK implantation. This effect of ENK was abolished by concomitant treatment with naloxone. Time-dependent changes in nuclear [3H]estradiol binding and the expression of estrogen and progesterone receptors were also detected in the hypothalamus of ENK-implanted and E2-treated rats. At 24-48 h following ENK implantation, expression of ERalpha and high affinity [3H]estradiol binding decreased. At this time point, the PR level was also reduced, while the ERbeta level was augmented. In conclusion, these results suggest that the stimulatory effects of E2 on the expression and activation of Akt protein and the expression of ERalpha and PR are negatively regulated in rat hypothalamus exposed to chronic ENK treatment.

Enkephalinergic involvement in substantia nigra in the modulation of hypothalamically-induced predatory attack behavior.

The present study was carried out in five cats which did not attack the rats spontaneously. Predatory attack on an anaesthetized rat was elicited by electrical stimulation of lateral hypothalamus at a mean current strength of 650 microA. The attack was accompanied by minimal affective display and culminated in neck biting. Microinfusions of DAME (delta-alanine methionine enkephaline) in 500 ng dose in substantia nigra facilitated the predatory attack and there was a significant reduction in the threshold current strength for affective display as well as somatomotor components. Microinfusions of naloxone, an opioid antagonist in 1.0 microg dose when DAME effect was at its peak reversed the facilitatory effects and the threshold returned to the control levels within 10 minutes of naloxone infusion at the same locus. Microinfusions of naloxone alone in similar dosage completely blocked the predatory attack response as indicated by an increase in the threshold current strength for somatomotor as well as affective display components. The somatomotor were completely inhibited and could not be elicited even when the current strength was increased to 1000 microA. Control injections of saline in similar volumes (0.5 microl) failed to produce any response Microinfusions of naloxone in lower dose (250 ng) failed to produce any blocking effect. These findings indicate that hypothalamically elicited predatory attack is facilitated by enkephalinergic mechanisms operating at the midbrain level.

Opioids suppress IPSCs in neurons of the rat medial septum/diagonal band of Broca: involvement of mu-opioid receptors and septohippocampal GABAergic neurons.

The medial septum/diagonal band region (MSDB), which provides a major cholinergic and GABAergic input to the hippocampus, expresses a high density of opioid receptors. Behaviorally, intraseptal injections of opioids produce deficits in spatial memory, however, little is known about the electrophysiological effects of opioids on MSDB neurons. Therefore, we investigated the electrophysiological effects of opioids on neurons of the MSDB using rat brain slices. In voltage-clamp recordings with patch electrodes, bath-applied met-enkephalin, a nonselective opioid receptor agonist, decreased the number of tetrodotoxin and bicuculline-sensitive inhibitory synaptic currents in cholinergic- and GABA-type MSDB neurons. A similar effect occurred in brain slices containing only the MSDB, suggesting that opioids decrease GABA release primarily by inhibiting spontaneously firing GABAergic neurons located within the MSDB. Accordingly, in extracellular recordings, opioid-sensitive, spontaneously firing neurons could be found within the MSDB. Additionally, in intracellular recordings a subpopulation of GABA-type neurons were directly inhibited by opioids. All effects of met-enkephalin were mimicked by a mu receptor agonist, but not by delta or kappa agonists. In antidromic activation studies, mu-opioids inhibited a subpopulation of septohippocampal neurons with high conduction velocity fibers, suggestive of thickly myelinated GABAergic fibers. Consistent with the electrophysiological findings, in double-immunolabeling studies, 20% of parvalbumin-containing septohippocampal GABA neurons colocalized the mu receptor, which at the ultrastructural level, was found to be associated with the neuronal cell membrane. Thus, opioids, via mu receptors, inhibit a subpopulation of MSDB GABAergic neurons that not only make local connections with both cholinergic and noncholinergic-type MSDB neurons, but also project to the hippocampus.

Electrostimulation of catecholamine release in the eel: modulation by antagonists and autocrine agonists.

The innervated chromaffin cells of the eel (Anguilla rostrata) release norepinephrine (NE) and epinephrine (E), while a component of the macrovascular wall releases dopamine (DA). The release of the three catecholamines is governed by complex controls which include adrenergic, nicotinergic, muscarinergic, and opioid mechanisms. To gain insight into the interactions between neural and autocrine factors in stimulated catecholamine release, we investigated the effect of adrenergic (phentolamine and propranolol) and muscarinergic (atropine) receptor antagonists, and of autocrine opioids (met-enkephalin, codeine, and morphine) on electrostimulated catecholamine secretion in situ. The hind brain (close to the root of nerve IX) of anesthetized eels was stimulated at four different time points, and segments of the posterior cardinal vein or the caudal vein were perfused with a saline solution, with or without test substances. Electrostimulation (30 s) four times within a total study duration of 14 min increased the release of DA, NE, and E into the perfusate of the cardinal vein. The vessel contains the innervated adrenomedullary equivalent. In the noninnervated caudal vein electrical stimulation had no impact on total DA release, while there was a slight decrease of NE release and a slight increase of E release. In the cardinal vein, both the alpha-adrenergic receptor antagonist phentolamine and the beta-adrenergic receptor antagonist propranolol strongly reduced the effect of electrostimulation on catecholamine release. Met-enkephalin reduced the release of all three catecholamines to a similar degree; its impact on NE release was especially strong. Codeine reduced the catecholamine release moderately, while morphine had no effect. Atropine reduced the release of all three catecholamines in a pattern similar to that of met-enkephalin. The findings on the posterior cardinal vein indicate that neurally stimulated NE and E release (1) involves autocrine/paracrine adrenergic mechanisms, (2) involves a muscarinergic mechanism, and possibly also endogenous codeine and morphine; and (3) is antagonized by met-enkephalin. The findings on the caudal vein are further evidence that macrovascular DA release is not under direct neural control.

Opioids suppress spontaneous and NMDA-induced inhibitory postsynaptic currents in the dorsal raphe nucleus of the rat in vitro.

Recently, local injection of morphine in the dorsal raphe nucleus (DRN) has been shown to increase serotonin release in the forebrain of unanesthetized rats. This study investigated the site of action of opioids in rat brain slices containing the DRN. Postsynaptic currents (PSCs), measured intracellularly under voltage clamp, were induced in serotonergic neurons with bath and microiontophoretic applications of NMDA to activate local neurons. Met-enkephalin (ENK) suppressed spontaneous and NMDA-induced GABAergic inhibitory PSCs. This effect, which was mimicked by the mu agonist DAMGO but not the kappa-agonist U50488 or the delta-agonist DPDPE, was reversed by the mu antagonist CTOP. ENK also suppressed spontaneous and NMDA-induced glutamatergic excitatory PSCs. By searching with focal microiontophoretic NMDA applications, GABAergic and glutamatergic cells projecting on serotonergic neurons were found in the DRN and the adjacent periaqueductal gray. Consistent with the reduction in PSCs, ENK inhibited/hyperpolarized the great majority (81%) of non-serotonergic neurons recorded extra- and intracellularly in the DRN; the ENK effect reversed polarity at -99 +/- 9 mV, close to the potassium reversal potential. In contrast, ENK inhibited/hyperpolarized only 28% of serotonergic neurons; in the affected cells, the ENK effect, blocked by CTOP, had its reversal potential shifted with change of extracellular potassium in agreement with the value predicted by the Nernst equation for a potassium conductance; serotonin occluded the ENK inhibition. Taken together, these results indicate that opioids inhibit both local GABAergic and glutamatergic cells projecting onto DRN serotonergic neurons.

Role of midbrain ventro-lateral tegmental area (VTA) enkephalinergic mechanisms in the facilitation of hypothalamically-induced predatory attack behaviour.

Bipolar concentric electrodes were implanted in five cats in extreme lateral regions of hypothalamus. These sites were electrically stimulated using biphasic square wave pulses at a current strength ranging from 300-800 microA to evoke predatory attack on an anaesthetized but live rat. At lower current strength (300 microA) only alertness with pupillary dilatation was produced. Gradual increase in the current strength led to the recruitment of somatic and affective components and a predatory attack was exhibited at a mean current strength of 700 microA. A scoring system allowed the construction of stimulus response curves, which remained fairly constant when repeated over a period of 3-4 weeks. Bilateral microinjections of delta-alanine methoinine enkephaline (DAME) (500 ng in 0.5 microliter saline) in ventrolateral tegmental area (VTA) elevated the mean threshold current strength for affective components while somatomotor components were totally inhibited. The blocking effect of DAME persisted for 1 hour. Microinjections of naloxone (1 microgram) in similar volumes facilitated the response as indicated by a reduction in threshold current strength for somatomotor and affective components. Microinjections of naloxone (1 microgram) in similar volumes facilitated the response as indicated by a reduction in threshold current strength for somatomotor and affective components. Microinjections of naloxone (1 microgram) also reversed the blocking effect of DAME and the thresholds returned to the control level within 10 min while microinjection of normal saline as control had no effect. The excitatory effects of naloxone and inhibitory effects of DAME were statistically significant at P < 0.01 and P < 0.05 respectively with Wilcoxon's signed rank test. The present study indicates that enkephalinergic as well as opioidergic mechanisms operating at the midbrain (VTA) level are involved in the inhibition of predatory attack as elicited from lateral hypothalamus.

Role of activation of calcium-sensitive K+ channels and cAMP in opioid-induced pial artery dilation.

The present study was designed to investigate the role of activation of Kca+2 channels and cAMP in opioid-induced pial artery dilation in newborn pigs equipped with closed cranial windows. Methionine enkephalin, an endogenous mu agonist, elicited dilation that was modestly attenuated by the Kca+2 channel antagonist, iberiotoxin (10(-7) M) (7 +/- 1, 11 +/- 1 and 16 +/- 1 vs. 4 +/- 1, 7 +/- 1, and 11 +/- 1% for methionine enkephalin 10(-10), 10(-8), 10(-6) M in the absence and presence of iberiotoxin, respectively). Dilator responses to leucine enkephalin and dynorphin, endogenous delta and kappa agonists, as well as the synthetic analogues DAMGO, DPDPE, deltorphin and U50488H all were similarly attenuated by iberiotoxin. Dilation in response to methionine enkephalin was accompanied by increased CSF cAMP concentration (1170 +/- 21, 1358 +/- 22, 1473 +/- 26, and 1575 +/- 24 fmol/ml for control, 10(-10), 10(-8), 10(-6) M methionine enkephalin, respectively). Methionine enkephalin-induced dilation was attenuated by Rp 8-bromo cAMPs (10(-5) M), a cAMP antagonist (7 +/- 1, 11 +/- 1 and 17 +/- 1 vs. 2 +/- 1, 4 +/- 1, and 7 +/- 1% for methionine enkephalin 10(-10), 10(-8), and 10(-6) M in the absence and presence of Rp 8-bromo cAMPs, respectively). Dilation by the other endogenous and synthetic opioid analogues was also accompanied by elevated CSF cAMP and attenuated by Rp 8-bromo cAMPs. Additionally, dilation produced by the cAMP analogue, 8-bromo cAMP, was blunted by iberiotoxin. These data show that both cAMP and activation of Kca+2 channels contribute to opioid-induced pial artery dilation. Further, these data suggest that opioids elicit dilation, at least in part, via the sequential release of cAMP and subsequent activation of Kca+2 channels by this second messenger.

Inhibitory role of opioid peptides in the regulation of aggressive and sexual behaviors in male Japanese quails.

We have recently isolated three opioid peptides, i.e., Met- and Leu-enkephalins and Met-enkephalin-Arg6-Phe7, from the avian brain. Furthermore, electrophysiological studies have shown that the dominant effect of these enkephalins on preoptic and hypothalamic neurons is an inhibition of neuronal activities in the male Japanese quail. The hypothalamus and preoptic area are known to be involved in the control of male reproductive behaviors, such as aggressive and sexual behaviors. To determine the functional role of opioid peptides in these reproductive behaviors, therefore, the present study was undertaken using adult males of the Japanese quail. We examined behavioral changes following an injection of naloxone (0.2, 2.0, and 20.0 nmol), a nonselective opioid receptor antagonist, or D-Ala2-Met5-enkephalinamide (DALA; 0.2, 2.0, and 20.0 nmol), a selective delta opioid receptor agonist, into the preoptic and anterior hypothalamic regions. Naloxone treatment showed a significant increase in the frequency of several aggressive actions and the effect was dose dependent. In contrast, DALA treatment significantly decreased the frequency of aggressive actions in a dose-dependent manner. Similar significant effects of these two drugs were observed in the sexual behavior. These findings provide the first evidence for the role of opioid peptides in the reproductive behaviors in the bird and suggest an inhibitory action of opioid to evoke the behaviors.

Stimulatory effect of intracerebroventricular met- and leu-enkephalin on corticosterone secretion in rats.

The significance of enkephalins for the function of the hypothalamic-pituitary-adrenal (HPA) axis, in spite of many efforts, is still elusive. We investigated the effect of leucine- and methionine-enkephalin on the HPA activity in conscious rats. These enkephalins, given intracerebroventricularly (i.c.v.) increased dose-dependently the activity of the HPA axis, measured indirectly through serum corticosterone levels. On a molar basis, leu-enkephalin exerted a stronger effect that met-enkephalin. Naloxone, an opioid receptor antagonist, given i.c.v. prior to enkephalins almost abolished the corticosterone response to met-enkephalin and significantly impaired the response to leu-enkephalin. Prazosin, an alpha 1-adrenergic antagonist, considerably reduced the increase in serum corticosterone levels induced by both enkephalins. Pretreatment of rats with yohimbine, an alpha 2-adrenergic antagonist, also considerably reduced the corticosterone response to met-enkephalin and significantly diminished the response induced by leu-enkephalin. Naloxone and yohimbine inhibited to the same extent the corticosterone response to met- and leu-enkephalin. This suggests an interaction between presynaptic opioid and alpha 2-receptors in regulation of the HPA function. Propranolol, a beta-adrenergic antagonist, given i.c.v. did not alter the corticosterone levels raised by met- and leu-enkephalin. These results indicate that both met- and leu-enkephalin increase the activity of the HPA axis in rats and both central opioid and adrenergic alpha-receptors are involved in this stimulation.

Predatory aggression induced by hypothalamic stimulation: modulation by midbrain periaqueductal gray (PAG).

Adequate electrical stimulation of extreme lateral hypothalamic regions of healthy, non-aggressive male cats was employed to produce aggression on live but anaesthetized rats. Stimulus response (S-R) curves based on scoring systems for both somatic and affective display components of behaviour were used to assess how manipulation of midbrain PAG by electrocoagulative lesions or drug microinjections affected the sensitivity of attack producing hypothalamic loci. Anodal lesions of dorsal PAG and adjoining tectum increased the excitability of hypothalamic loci producing predatory attack. Microinjection of 250 ng of delta-alanine-methionine enkephalin (DAME) in dPAG completely suppressed the somatomotor components of attack behaviour and markedly inhibited the affective display components. Administration of naloxone, an opioid antagonist (1 microgram) at the same sites facilitated the hypothalamically induced attack behaviour and annulled the inhibitory effect of DAME. These findings indicate the involvement of midbrain enkephalinergic mechanisms in the modulation of predatory attack behaviour elaborated by hypothalamic stimulation.

Effects of a mixture of peptidase inhibitors (amastatin, captopril and phosphoramidon) on Met-enkephalin-, beta-endorphin-, dynorphin-(1-13)- and electroacupuncture-induced antinociception in rats.

The effects of a mixture of three peptidase inhibitors (PIs), amastatin, captopril and phosphoramidon, on methionine-enkephalin (Met-enk)-, beta-endorphin (beta-end)-, dynorphin-(1-13) (Dyn)- and electroacupuncture (EA)-induced antinociception were compared in rats. EA was performed by passing electric pulses (3 Hz, 0.1-msec duration, for 45 min) through acupuncture needles inserted into the Hoku-point. The antinociceptive effect was estimated by the hind paw pressure test. The antinociceptive effects of Met-enk and beta-end injected i.c.v. or i.t. and of Dyn injected i.t. were clearly potentiated by the PIs pretreated by the same administration routes as used for the injection of opioid peptides. The antinociceptive effects of Met-enk, beta-end and Dyn injected i.c.v. were also potentiated significantly by i.t.-PIs. PIs injected into the periaqueductal gray (PAG) potentiated EA antinociception. However, the EA effect was not affected by i.t.-PIs and was rather attenuated by i.c.v.-PIs. These results suggest that: i) Met-enk hydrolyzing enzymes are involved in the degradation of not only Met-enk but also beta-end and Dyn in the rat central nervous system; ii) Met-enk and beta-end act on both supraspinal and spinal sites, while Dyn acts only on the spinal site; iii) EA antinociception is mediated by supraspinal Met-enk and/or beta-end; and iv) an anti-opiate peptide system may be activated by EA stimulation, being susceptible to Met-enk hydrolyzing enzymes.

Differentiation of intracranial morphine self-administration behavior among five brain regions in mice.

BALB/c mice were unilaterally implanted with a guide cannula, the tip of which was positioned 1.5 mm above either the lateral hypothalamus (LH) the medial hypothalamus (MH), the mesencephalic central gray area (CG), or either the dorsal (DRF) or ventral parts (VRF) of the reticular formation. On each day of the experimental period a stainless steel injection cannula was inserted into these brain structures to compare the self-administration of two doses of morphine (5 ng or 50 ng), using a spatial discrimination task in a Y-maze. At the dose of 5 ng, LH-, MH-, CG-, and VRF-injected mice all showed a regular self-administration response. At the dose of 50 ng, a discrimination between the reinforced arm and the neutral arm of the Y-maze was observed in LH-, MH-, and VRF-injected mice. Animals of the MH group exhibited the highest level of discrimination performance. At this dose, long injection latencies (> 15 min) were recorded in the CG group, which constrained us to reduce the number of daily trials from 10 to 4. In these modified conditions, CG animals clearly self-injected the dose of 50 ng of morphine. Subcutaneous injections of naloxone (4 mg/kg) reduced the number of self-administrations of morphine at each of the four responding structures. Marked signs of physical dependence (escape attempts) were observed in the four groups but with a higher frequency in CG and MH animals. When the injections of naloxone were suspended, a regular self-administration reappeared.(ABSTRACT TRUNCATED AT 250 WORDS)

Antitussive effect of [Met5]enkephalin-Arg6-Phe7 in mice.

We examined the effect of [Met5]enkephalin-Arg6-Phe7 (MEAP) on the capsaicin-induced cough reflex in mice. Intracerebroventricular administration of MEAP significantly decreased the number of coughs in a dose-dependent manner. The antitussive effect of MEAP was blocked by nor-binaltorphimine, a selective kappa-opioid receptor antagonist. However, beta-funaltrexamine, a mu-opioid receptor antagonist, had no effect on the antitussive effect of MEAP. On the other hand, the antinociceptive effect of MEAP, as determined in the tail-flick test, was blocked by both nor-binaltorphimine and beta-funaltrexamine. Naltrindole, a delta-opioid receptor antagonist, had no effect on either the antitussive effect or the antinociceptive effect of MEAP. These data suggest that MEAP exerts its antitussive effect in mice through the stimulation of kappa-opioid receptors, whereas the antinociceptive effect of MEAP is mediated through the simulation of both kappa- and mu-opioid receptors.

[The effect of Leu- and Met-enkephalins on the adenosine deaminase and 5'-nucleotidase activities of the lymphocytes under conditions of stress stimulation of metastasis]. / Vliianie lei- i met-énkefalinov na aktivnost' adenozindezaminazy i 5'-nukleotidazy limfotsitov v usloviiakh stressornoi stimuliatsii metastazirovaniia.

A correlation between changes of the hypothalamic mediator processes, hormone level in blood plasma, catecholamines and their catabolites in urine and changes of the activity of adenosine metabolism enzymes in the thymus and the spleen lymphocytes has been observed in C57B1 mice with Lewis lung carcinoma. Effect of leu- and met-enkephalines on neuroendocrine stress-realizing mechanisms proved to be different, that is responsible for their contrary effect on the metastatic growth--leu-enkephaline inhibits and met-enkephaline stimulates the process. It has been shown that an inhibitory effect of leu-enkephaline is greatly related to its stimulating influence on the thymus lymphocyte activity and a decrease of the functional activity of the spleen lymphocytes.

Opioid peptide regulation of neurons in the bed nucleus of the stria terminalis: a microiontophoretic study.

Single unit activity was recorded from neurons in the bed nucleus of the stria terminalis (BNST) in response to single or combined microiontophoretic ejection of D-Ala2-Met5-enkephalinamide (DAME) and naloxone. BNST neurons showed predominantly excitatory responses following small iontophoretic applications of DAME, and these responses were antagonized by naloxone. In contrast, inhibitory responses that were elicited by DAME required larger ejection currents and usually failed to show naloxone antagonism. The results indicate that activation of enkephalin-sensitive BNST neurons by DAME is primarily, but not exclusively, excitatory, which suggests that these responses may reflect differences in receptor sensitivity to the applied agonist.

Effect of D ala2 metenkephalinamide on feline jejunal and ileal water and electrolyte transport.

The aim of this investigation was to compare the effect of an opioid, D ala2 metenkephalinamide (DAMA), on net jejunal and ileal water and electrolyte fluxes using the gut perfusion technique in the anesthetized cat. Intestinal transport was measured during intravenous infusion of serial doses of 2, 6, and 18 micrograms.kg-1.h-1 of DAMA in 6 cats. Each cat was its own control during an intravenous infusion of 150 mmol/l NaCl preceding the first dose of peptide and following the last dose of DAMA. Both jejunal and ileal segments were isolated by inflated balloons and were studied at the same time. Fifteen ml of an iso-osmolar test solution with hypo-osmolar ion contents and complementary mannitol were administered in the upstream tube and collected 1 h later in the downstream tube. In the jejunum, water secretion was dose-dependently reversed to an absorption from a control value of +0.5 +/- 0.4 to -0.83 +/- 0.5 ml.h-1.10 cm-1; in the ileum, water absorption was increased from -0.5 +/- 0.3 to -1.5 +/- 0.2 ml.h-1.10 cm-1. The net absorption of all electrolytes, ie sodium, chloride, bicarbonate, potassium and calcium also increased during peptide administration. However, a qualitative difference in the ion transport was observed between the jejunum and the ileum.