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.

Production and characterization of antibodies for the specific determination of the opioid peptide Met5-Enkephalin-Arg6-Phe7.

Endogenous opioids serve as modulators of neuroendocrine and immune system processes, the investigation of which calls for high-specificity radioimmunoassays (RIAs). This study focuses on the development and use of a specific radioimmunoassay for the opioid peptide Met5-Enkephalin-Arg6-Phe7 (MEAP), the C-terminus part of proenkephalin A. Antibodies were raised in four rabbits and investigated in terms of titre, avidity and specificity, followed by finding ideal conditions for these antibodies in RIA. MEAP concentrations were determined in crude extracts of rat hypothalamus, dorsal root ganglia, adrenals and ankle using this standardized assay after an oxidizing process. At reverse-phase high-pressure liquid chromatography (HPLC), the position of immunoreactive material from rat hypothalamus eluted as two peaks out of which one was compatible with that of synthetic MEAP. All rabbits exhibited individual differences in relative immune response and time of its onset. The avidity constant was 10 times higher on a molar basis for ab 4108 compared with ab 4182. There was no cross-reactivity for ab 4182 to related peptides, such as enkephalins and dynorphin B, and negligible background values for ab 4108. The relative levels ofimmunoreactive MEAP from the CNS versus peripheral tissues contrasted in accordance with current knowledge. It is suggested that reports with RIA results should include characterization of antibodies, extraction procedures, standard curves and compositions of buffers. Furthermore, the results should preferably be expressed in relation to total protein content.

Immunocytochemical distribution of Met-enkephalin-Arg6-Gly7-Leu8 (Met-8) in the auditory system of the rat.

Methionine-enkephalin-Arg(6)-Gly(7)-Leu(8) (Met(8)) is known to act as a neurotransmitter or neuromodulator and it has been implicated in pain, cardiovascular and motor mechanisms, but its role in audition is currently unknown. In the present study we have applied an immunocytochemical technique and describe the distribution of cell bodies and fibers containing Met(8) in the auditory pathway of the rat. The main finding is that we found either Met(8)-immunoreactive fibers or cell bodies or both in virtually all nuclei of the rat auditory system except for the medial superior olive and the ventral division of the medial geniculate body in which we did not find any immunoreactivity for Met(8). This suggests that the neuropeptide Met(8) is widely distributed throughout the auditory system of the rat. Our results suggest that Met(8) could play at least two roles in hearing. It seems to be involved in the processing of the descending auditory pathway, and it may be implicated in the multisensory integration of auditory information that takes place in the non-lemniscal auditory pathway.

The effect on opioid peptides in the rat brain, after chronic treatment with the anabolic androgenic steroid, nandrolone decanoate.

In recent years, an increase in abuse of anabolic androgenic steroids (AAS) has been seen among individuals not directly connected to sports. Clinical evidence suggests that abuse of these steroids may result in profound changes in personality, expressed by depressive symptoms, irritability and increased aggression. It is still unknown whether these alterations are related to changes in any particular transmitter system or whether they are persistent or reversible. In this study we focused on AAS effect on the endogenous dynorphin and enkephalin system in the brain. Male rats were given intramuscular injections of the AAS nandrolone decanoate (15 mg/kg), once daily for 2 weeks. The levels of the opioid peptide immunoreactivities (ir) were assessed by radioimmunoassay in two groups immediately after the treatment and in two other groups after additional 3 weeks without any drug treatment (recovery period). The result indicates that chronic AAS treatment increased the activity in the dynorphin B- and Met-enkephalin-Arg(6)Phe(7)-ir in the hypothalamus, striatum and periaqueductal gray (PAG) compared to controls. In addition, the steroid induced an imbalance between the dynorphin and the enkephalin opioid system in the nucleus accumbens, hypothalamus and PAG. This imbalance remained after the recovery period. Since increased peptide activity was found in brain regions regulating emotions, dependence, defensive reactions and aggression, it was suggested that the actual endogenous opioid systems are involved in previously reported AAS-induced changes in these behaviours.

[Modulation of the antiarrhythmic effect by endogenous opioids during adaptation to hypoxia in rats]. / O moduliruiushchem vliianii éndogennykh opioidov na antiaritmicheskii éffect pri adaptatsii krys k gipoksii.

Adaptation of rats to hypoxia increased the heart resistance against arrhythmogenic effects. The Met-enkephaline-Arg6-Phe7 contant increased in the hypothalamus of the adapted animals. The blockers of mu- and delta-opioid receptors reduced the antiarrhythmic effect of the adaptation, whereas chi-receptor inhibitor, nor-binaltorphimine, completely abolished it. The findings suggest the antiarrhythmic effect depends on an increase of the endogenous opioids level and modulated effect of these peptides upon autonomous nervous system.

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.

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.

Enzymatic peptide synthesis in low water content systems: preparative enzymatic synthesis of [Leu]- and [Met]-enkephalin derivatives.

A novel total enzymatic synthesis of [Leu]- and [Met]-enkephalin derivatives was accomplished in low-water content systems at a preparative scale. alpha-Chymotrypsin, papain, thermolysin and bromelain adsorbed on Celite were used as catalysts. Organic solvents such as acetonitrile and ethyl acetate with small amounts of buffer added or at specific water activity were used as reaction media. Simple readily available amino acid ester derivatives were used as starting building blocks. This feature allowed the possibility of using the products in one step directly as acyl-donor ester, without any chemical or enzymatic modification, in the next enzymatic coupling. The optimal strategy for the synthesis of the enkephalin derivatives was different depending on the carboxy terminal group. The preparation of the carboxy-terminal amide derivatives (R-Tyr-Gly-Gly-Phe-Leu[Met]-NH2) was achieved via 4 + 1 fragment condensation catalyzed by alpha-chymotrypsin. The carboxy-terminal ethyl ester derivatives (R-Tyr-Gly-Gly-Phe-Leu[Met]-OEt) were obtained via 2 + 3 condensation catalyzed by bromelain, a quite unusual protease for peptide synthesis but more effective than papain in this coupling. Both syntheses were carried out in four enzymatic steps and one or two chemical deprotection steps routinely used in peptide synthesis. The overall yields of pentapeptide derivatives were between 40-54% of pure product.

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.

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.

Direct and indirect enkephalinergic synaptic inputs to the rat arcuate nucleus studied by combination of retrograde tracing and immunocytochemistry.

The origin of both direct and indirect enkephalinergic innervation potentially able to influence neurons of the rat arcuate nucleus has been investigated by combining enkephalin immunocytochemistry and retrograde axonal transport of a wheatgerm agglutinin-Apo horseradish peroxidase-gold complex. Twenty four hours after tissue injections of small volumes (20 nl) of the tracer into the arcuate nucleus, rats were treated with colchicine and killed. In order to localize the enkephalinergic cells which directly innervate the arcuate nucleus, Vibratome sections were first silver-stained for detection of the wheatgerm agglutinin-Apohorseradish peroxidase-gold complex and then processed for enkephalin immunohistochemistry. To study the indirect enkephalinergic input to the arcuate nucleus, an electron microscope detection of immunoreactive synapses was carried out in areas rich in retrogradely labeled perikarya. Perikarya both immunoreactive and retrogradely labeled were observed ipsilaterally to the injection site in telencephalic structures such as the bed nucleus of the stria terminalis, medial preoptic and adjacent periventricular areas. Hypothalamic ipsilateral doubly labeled cells were localized principally in the dorsomedial nucleus and rostral arcuate nucleus. The major direct inputs arising from brainstem structures concerns the dorsal and ventral parabrachial nuclei. Moreover, at the ultrastructural level, numerous enkephalinergic terminals were demonstrated to synapse with retrogradely labeled perikarya and dendrites localized in the medial preoptic area, the hypothalamic paraventricular nucleus and the parabrachial nuclei providing evidence for an important enkephalinergic input on neurons projecting to the arcuate nucleus. Taken together, our light and electron microscope studies strongly suggest that the arcuate nucleus is the target of an enkephalinergic control originating from several regions and acting either directly or indirectly on neurons projecting to the arcuate nucleus.

Effects of hypnosis on plasma proenkephalin peptide F and perceptual and cardiovascular responses during submaximal exercise.

Little information is available concerning the influence of subconscious mechanisms on neuroendocrine function, more specifically, proenkephalin peptide F release. Ten men [5 middle distance runners (21.6 (SD 0.54 years) and 5 untrained men (24.0 (SD 4.3 years)] consented to be volunteers in this investigation. Submaximal exercise intensities of 25% and 50% of peak oxygen consumption (VO2) (8 min stages) were used for both the control and hypnosis treatments. A traditional hypnotic induction was used, with the suggestion of two higher intensities of exercise stress (50% and 75% peak VO2) previously experienced in familiarization and testing by each subject. Each minute oxygen consumption was measured using open circuit spirometry, heart rate via an ECG, and ratings of perceived exertion (RPE) using the Borg scale. Plasma peptide F immunoreactivity (ir) [preproenkephalin-(107-140)] in blood sampled from an indwelling cannula was measured by radioimmunoassay at 7-8 min of each stage of the exercise test. Expected significant increases were observed for all cardiorespiratory and perceptual variables over the increasing exercise intensities and there were no significant differences between trained and untrained groups for peptide F if response patterns. Hypnosis did not significantly affect peptide F ir concentrations (P > 0.05) and did not significantly alter exercise heart rate, RPE or minute ventilation (P > 0.05). However, hypnosis did significantly increase oxygen consumption during exercise (P = 0.0095) but not of the magnitude needed for the metabolic demands of the higher exercise intensities. Thus, traditional hypnosis was unable to make functionally significant changes in the cardiorespiratory variables.(ABSTRACT TRUNCATED AT 250 WORDS)

[The participation of prostanoids in the realization of the cardioprotective, antistressor and anti-arrhythmic effects of enkephalins]. / Uchastie prostanoidov v realizatsii kardioprotektornykh, antistressornykh i antiaritmicheskikh éffektov énkefalinov.

It has been established that preliminary administration of D-Ala2-Leu5-Arg6-enkephalin, D-Met2-Pro5-enkephalinamide, and D-phenylalanine to experimental animals prevents a stress-induced increase of the content of thromboxane in the blood plasma and myocardium and induces a rise of the level of prostacyclin, PGF2 and PGE in the heart and blood plasma. The authors hold that the changes in the level of prostanoids may mediate cardioprotective and anti-stressor rather than antiarrhythmic effects of enkephalins.

Effect of low- and high-frequency TENS on Met-enkephalin-Arg-Phe and dynorphin A immunoreactivity in human lumbar CSF.

Transcutaneous nerve stimulation (TENS) treatment was given for 30 min to 37 patients divided into 3 groups of 10 patients and 1 group of 7 patients. Two groups received low-frequency (2 Hz) and the other 2 groups high-frequency (100 Hz) stimulation. A diagnostic lumbar cerebrospinal fluid (CSF) sample was obtained immediately before and after stimulation. The CSF samples were subjected to analysis of immunoreactive (ir) opioid peptides, Met-enkephalin-Arg-Phe (MEAP) from preproenkephalin and dynorphin A (Dyn A) from preprodynorphin, respectively. Low frequency TENS applied on the hand and the leg resulted in a marked increase (367%, P less than 0.05) of ir-MEAP but not ir-Dyn A, whereas high-frequency (100 Hz) TENS produced a 49% increase in ir-Dyn A (P less than 0.01) but not ir-MEAP. This is the first report in humans that 2 Hz and 100 Hz peripheral stimulation induces differential release of peptides from preproenkephalin and preprodynorphin, respectively.

SCPB-and FMRFamide-like immunoreactivities in lobster neurons: colocalization of distinct peptides or colabeling of the same peptide(s)?

Virtually all of the SCPB-like immunoreactive neurons (ca. 60 cells) in the lobster Homarus americanus also contain FMRFamide-like immunoreactivity. Control experiments reveal that SCPB-and FMRFamide-like immunoreactivities are successfully preadsorbed with their specific antigens, while the normal staining pattern is retained following preadsorption of each antibody with the alternate peptide. These experiments potentially lead to the conclusion that the anti-SCPB and anti-FMRFamide antibodies are labeling distinct compounds that are colocalized in lobster neurons. The lobster nervous system does not, however, contain authentic FMRFamide, but rather several FMRFamide-like compounds (Trimmer et al., J. Comp. Neurol. 266:16-26, 1987). The most abundant of these is the octapeptide TNRNFLRFamide. Experiments demonstrate that SCPB-like immunoreactivity is completely preadsorbed with synthetic TNRNFLRFamide, while there is a significant or complete loss of staining after preadsorption of the FMRFamide antibody with this molecule. Met-enkephalin-Arg-Phe-amide (YGGFMRFamide), an extended opioid peptide containing the FMRFamide sequence, also preadsorbs SCPB- and FMRFamide-like immunoreactivities, while Met-enkephalin-Arg-Phe (YGGFMRF) has no effect on the staining properties of these antibodies. These results suggest that the SCPB antibody can bind to extended forms of FMRFamide-like molecules, and that anti-SCPB and anti-FMRFamide antibodies may be colabeling one or more FMRFamide-like molecules in lobster neurons.

The effect of single and repeated ethanol administration of hypothalamic opioid systems activity --an in vitro release study.

The effect of ethanol administration on the in vitro release of alpha-neoendorphin (ANEO) and Met-enkephalin-Arg6-Gly7-Leu8 (MEAGL), peptides derived from prodynorphin and proenkephalin, respectively, was studied in the rat hypothalamus. Single ethanol administration had no effect on the tissue level and potassium-stimulated release of these peptides. Repeated ethanol administration increased the release of ANEO and reduced the release of MEAGL from hypothalamic slices. Two days after cessation of ethanol administration the release of MEAGL returned to control values, whereas the release of ANEO was significantly inhibited. On the other hand, the tissue level of those peptides remained unchanged after repeated ethanol or 2 days after its last administration. The present study shows that repeated treatment with ethanol may lead to an increase in the prodynorphin neurons' sensitivity to the depolarizing effect of potassium. In contrast, the sensitivity of the proenkephalin system to ethanol seems to be inhibited. Thus, ethanol appears to have an opposite effect on the sensitivity of hypothalamic proenkephalin and prodynorphin neurons.

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.