Differential metabolism of dynorphins in substantia nigra, striatum, and hippocampus

To map the proteolytic enzymes metabolizing dynorphins in brain structures, size-exclusion chromatography linked to electrospray ionization mass spectrometry was used. Enzymes extracted from rat hippocampus, striatum, and substantia nigra were tested for their capability of converting dynorphin-related peptides. Dynorphin A was the most resistant to proteolytic conversion, whereas Big dynorphin and dynorphin B-29 were slowly converted to dynorphin A and dynorphins A and B, respectively. Dynorphin B and alpha-neoendorphin were the least resistant. Dynorphin B was rapidly converted to Leu-enkephalin in the striatum and hippocampus but to Leu-enkephalin-Arg6 in the substantia nigra. alpha-Neoendorphin was converted to Leu-enkephalin in all tissues investigated.     

Release into ventriculo-cisternal perfusate of beta-endorphin- and Met-enkephalin-immunoreactivity: effects of electrical stimulation in the arcuate nucleus and periaqueductal gray of the rat

To examine the resting and evoked release of the endogenous opioid peptides beta-endorphin and Met-enkephalin from brain, we examined the levels of the respective immunoreactivities in the lateral ventricle-cisterna magna perfusate of the halothane-anesthetized rat. Ten Hz but not 100 Hz stimulation in the arcuate nucleus (ARC) of the hypothalamus released beta-endorphin immunoreactivity (beta-EPir) to the perfusate, whereas 100 Hz but not 10 Hz stimulation in the periaqueductal gray (PAG) of the mid brain released Met-enkephalin immunoreactivity (MEir). MEir was not released by stimulation in ARC and beta-EPir was not released by stimulation in PAG. Characterization of the released beta-EPir and MEir by high performance liquid chromatography showed that authentic beta-endorphin and Met-enkephalin were the major constituents of beta-EPir and MEir, respectively. Systemic administration of the dopaminergic antagonist haloperidol increased plasma, but not perfusate levels of beta-EPir. Both the opioid antagonist naloxone and the NMDA antagonist MK-801 failed to affect beta-EPir or MEir release. ARC and PAG stimulated inhibited a nociceptive reflex (tail-dip in 52.5 degrees C water), and naloxone did not reliably reverse this inhibition. These data support the previously suggested possibility of opioid mediation of stimulation induced analgesia, although we were unable to confirm the theory by naloxone reversibility in this study. Furthermore, the data support the assumption that measurement of opioid peptides in cerebrospinal fluid is a relevant approach in research aimed at elucidating the physiological and pathophysiological roles of endogenous opioid peptides.     

Memory T cell tolerance to superantigens is not due to increased susceptibility to apoptosis

Opioid peptides were analysed in tissue extracts of various brain structures and the pituitary gland from rats sacrificed by microwave irradiation, and compared with peptide levels in tissue extracts from decapitated rats. Dynorphin A, dynorphin B and Leu-enkephalinArg6, derived from prodynorphin, and Met-enkephalinArg6Phe7 from proenkephalin, were measured. Basal immunoreactive levels of dynorphin A and B were consistently higher in extracts from microwave-irradiated rats, whereas in these extracts immunoreactive levels of Leu-enkephalinArg6, an endogenous metabolite of dynorphin peptides, were either lower than, the same as or higher than in decapitated rats. Immunoreactive levels of Met-enkephalinArg6Phe7 were higher in microwave-irradiated rats. Effects of morphine treatment on prodynorphin peptide levels were evaluated and compared with previous findings in decapitated rats. Dynorphin immunoreactive levels were higher in the nucleus accumbens and striatum of morphine-tolerant rats than in corresponding areas in saline-treated rats. These results indicate tissue-specific metabolism of prodynorphin peptides and show that metabolism of opioid peptides occurs during the dissection procedure after decapitation of the rat even though precautions are taken to minimize degradation.     

Hypothalamic Leu-enkephalin-immunoreactive fibers terminate on calbindin-containing somatospiny cells in the lateral septal area of the rat

Correlated light and electron microscopic double-immunostaining experiments for Leu-enkephalin and calbindin were employed to determine the postsynaptic targets in the septal complex of Leu-enkephalin fibers. Chronic surgical isolation of the septal complex from its hypothalamic afferents and retrograde tracer studies using wheat germ agglutinin-conjugated horseradish peroxidase, both followed by an immunostaining for Leu-enkephalin, were performed to elucidate the location of the origin of these axon terminals. Furthermore, a colocalization study for glutamic acid decarboxylase and Leu-enkephalin was carried out on hypothalamic sections to determine their possible coexistence in cells projecting to the lateral septum. These studies revealed that 1) Leu-enkephalin-immunoreactive axons form pericellular baskets around a population of lateral septal area neurons; 2) they establish exclusively asymmetric synaptic contacts on their soma and initial dendritic segments; 3) 10% of the lateral septal area calbindin-containing cells, which are all of the gamma-aminobutyric acid (GABA)-ergic somatospiny type, are innervated by Leu-enkephalin-immunoreactive baskets; 4) only 40% of the Leu-enkephalin target neurons are calbindin immunopositive; 5) the septopetal Leu-enkephalin fibers derive from neurons located in the ipsilateral perifornical area and anterior hypothalamus; and 6) none of their cells of origin cocontains the inhibitory transmitter GABA. These observations indicate that hypothalamic Leu-enkephalin-containing neurons are non-GABAergic excitatory cells. Hence, they can effectively stimulate a population of lateral septal area neurons, including the somatospiny cells, which are all GABAergic. Therefore, after stimulatory Leu-enkephalin action, these neurons can inhibit their postsynaptic targets, including other projective lateral septal neurons.     

Initial processing of human proenkephalin in bovine chromaffin cells

The opioid peptide precursor preproenkephalin (PPE) contains seven enkephalin sequences and is synthesized by epinephrine-producing adrenal chromaffin cells and various peripheral and central neurons. After removal of its signal peptide, PPE undergoes processing at dibasic amino acid sites to yield its final opioid products-Met-enkephalin, Leu-enkephalin, and various larger, enkephalin-containing peptides. Processing of PPE was examined in bovine chromaffin cells using a plasmid containing the human PPE (hPPE) cDNA under the control of the cytomegalovirus immediate early enhancer/promoter. Following transfection of this hPPE-containing plasmid into bovine chromaffin cells, several proenkephalin-immunoreactive bands were observed on western blots with monoclonal antibodies that recognize human, but not bovine, proenkephalin sequences. The pattern of hPPE-derived peptides observed was similar to that of bovine PPE processing products. A series of recombinant plasmids containing mutations in the hPPE sequence at putative processing sites was then constructed. Conversion of Lys-Lys and Lys-Arg sequences to Lys-Gln and of Arg-Arg to Arg-Gln altered initial hPPE processing at only three of the putative processing sites. When hPPE cDNA containing mutations at all of these initially processed sites was expressed, one or more alternative processing sites were revealed. These data suggest the importance of structural features in addition to the dibasic sequences that limit the processing of proenkephalin.     

Differential effects of beta-endorphin and Met- and Leu-enkephalin on steroid hormone-induced lordosis in ovariectomized female rats

The effect of intrathirdventricular (I.T.V.) injections of beta-endorphin, anti-beta-endorphin antiserum, Met-enkephalin, Leu-enkephalin, and naloxone on the initial activation and final development of steroid hormone-mediated induction of female sexual receptivity was studied in ovariectomized female rats. The lordosis response to male mounts in ovariectomized rats after subcutaneous (S.C.) estradiol benzoate (EB) and progesterone (Prog) priming was facilitated by beta-endorphin, and Met-enkephalin (10 microg x 5 microl(-1)), but inhibited by Leu-enkephalin, when the peptides were injected into the third ventricle at the time of S.C. EB priming. A lower dose Met-enkephalin had no effects. Lordosis behavior in steroid hormone-primed rats was significantly facilitated when I.T.V. injections of Met-enkephalin were given 1 h prior to behavioral testing (47 h after EB priming). At 1 h prior to behavioral testing (47 h after EB priming), I.T.V. injection of beta-endorphin significantly inhibited lordosis behavior, especially at the higher dose of beta-endorphin (10 microg x 5 microl(-1)). Under those conditions, Leu-enkephalin had no effect. Lordosis behavior of ovariectomized female rats receiving S.C. steroid hormones and I.T.V. injection of anti-beta-endorphin antiserum was significantly inhibited when anti-beta-endorphin antiserum was injected at the time of EB priming. However, lordosis was significantly facilitated when anti-beta-endorphin antiserum was injected 1 h prior to the behavior testing (47 h after EB priming). In contrast, I.T.V. injection of the opioid antagonist naloxone given either at the time of EB priming or 1 h prior to behavioral testing (47 h after EB priming) decreased lordosis behavior. The present results suggest that 1) beta-endorphin, Met-enkephalin, and Leu-enkephalin have differential effects in the control of lordosis behavior; 2) the opioidergic systems may modulate initial-stage and final-stage estrogen-induced lordosis behavior; and 3) the opioidergic systems could be divided into the endorphinergic modulation-type and enkephalinergic modulation-type, based on their effects on lordosis behavior.     

Met-enkephalin Arg-Phe-immunoreactive neurons in the central nervous system of the pond snail Lymnaea stagnalis

The distribution of an opioid peptide related to YGGFMRF was determined in the CNS and other organs of the pond snail, Lymnaea stagnalis, by RIA and immunocytochemistry. RIA revealed the highest levels in the CNS (1 pmol/organ) and penis (400 fmol/organ). There were also significant levels in the haemolymph, most of which was not associated with haemocytes (580 fmol/ml). Both serial section and whole-mount immunocytochemistry of the CNS revealed immunoreactive cells in every ganglion with the majority in the cerebral and pedal ganglia. In the pedal ganglia some of the immunoreactive cells were close to the cells of the A-cluster, which are known to respond to opioids, and could innervate them. In the cerebral ganglia the immunoreactive cells included a group of neurosecretory cells, the caudo dorsal cells (CDCs) and the terminals of these cells in the cerebral commissure were also stained. The CDCs secrete peptides into the haemolymph and so could be the source of the YGGFMRF immunoreactivity. Immunoreactivity (including the CDCs) was observed in locations that correspond to those reported for other fragments of proenkephalin, such as Met- and Leu-enkephalin, suggesting that they may share a common precursor, a Lymnaea proenkephalin. A map of the 358 YGGFMRF-immunoreactive cells in the CNS is presented, many of which have not been previously identified.     

Expression of the human antigen SPAG2 in the testis and localization to the outer dense fibers in spermatozoa

There is increasing evidence to suggest that opioid peptides may have widespread effects as regulators of growth. To evaluate the hypothesis that endogenous opioids control cellular proliferation during neural development, we have used in situ hybridization to examine opioid peptide and receptor mRNA expression in neuroepithelial zones of fetal rat brain and spinal cord. Our data show that proenkephalin mRNA is widely expressed in forebrain germinal zones and choroid plexus during the second half of gestation. In contrast, prodynorphin mRNA expression is restricted to the periventricular region of the ventral spinal cord. Little mu or delta receptor mRNA expression was detected in any regions of neuronal proliferation prior to birth. However, kappa receptor mRNA is widely expressed in hindbrain germinal zones during the 3rd week of gestation. Our present findings support the hypothesis that endogenous opioids may regulate proliferation of both neuronal and non-neuronal cells during central nervous system development. Given the segregated expression of proenkephalin mRNA in forebrain neuroepithelium and kappa receptor mRNA within hindbrain, different opioid mechanisms may regulate cell division in rostral and caudal brain regions.     

Effect of enkephalins and endorphins on cytotoxic activity of natural killer cells and macrophages/monocytes in mice

The effect of [Met5]enkephalin, [Leu5]enkephalin, proenkephalin, dynorphin-(1-17) or beta-endorphin on the cytotoxic (51Cr release assay) activity of natural killer cells and macrophages/monocytes was studied in mice. It was found that a single i.p. injection of [Met5]enkephalin, [Leu5]enkephalin, beta-endorphin, dynorphin or proenkephalin as well as repeated treatment with both enkephalins increased natural killer cell activity. In vitro only [Met5]enkephalin stimulated natural killer cells. Opioid peptides did not affect the cytotoxic activity of macrophages/monocytes. In addition to functional alterations, both enkephalins and beta-endorphin increased the percentage of cells with natural killer phenotype. The results of this study suggest that the increase in natural killer cytotoxicity after opioid peptides injected once or for 14 days may result from an increased number of natural killer cells in the spleen.     

Methamphetamine alters prodynorphin gene expression and dynorphin A levels in rat hypothalamus

Chronic administration of morphine or cocaine affects opioid gene expression. To better understand the possible existence of common neuronal pathways shared by different classes of drugs of abuse, we studied the effects of methamphetamine on the gene expression of the opioid precursor prodynorphin and on the levels of peptide dynorphin A in the rat brain. Acute (6 mg/kg, intraperitoneally, i.p.) and chronic (6 mg/kg, i.p. for 15 days) methamphetamine markedly raised prodynorphin mRNA levels in the hypothalamus, whereas no effect was observed in the hippocampus. Dynorphin A levels increased after chronic treatment in the hypothalamus and in the striatum, whereas no significant changes were detected after acute treatment. These results indicate that methamphetamine affects prodynorphin gene expression in the hypothalamus, which may be an important site (also for its relevant neuroendocrine correlates) for opioidergic mechanisms activated by addictive drugs.     

Nanoparticle technology for delivery of drugs across the blood-brain barrier

The Leu-enkephalin dalargin and the Met-enkephalin kyotorphin normally do not cross the blood-brain barrier (BBB) when given systemically. To transport these neuropeptides across the BBB they were adsorbed onto the surface of poly(butylcyanoacrylate) nanoparticles (NPs) and the NPs were coated with polysorbate 80. Central analgesia was measured by the hot plate test in mice. The antidepressant amitriptyline, which normally penetrates the BBB, was used to examine the versatility of the NP method. The concentration of amitriptyline in serum and brain of mice was determined by a gas chromatographic method. Furthermore, NPs were fabricated with different stabilizers. After the adsorption of the peptides on polysorbate 85-stabilized NPs, analgesia was noted after intravenous application when NPs were not coated. The amitriptyline level was significantly enhanced in brain when the substance was adsorbed onto the NP and coated or when the particles were stabilized with polysorbate 85.     

The corticostriatal system mediates the "paradoxical" contraversive rotation but not the striatal hyperexpression of Fos induced by amphetamine early after 6-hydroxydopamine lesion of the nigrostriatal pathway

Opioid peptides are known to play a role in the function and growth of the mammalian heart. Although some information about gene expression of opioids in the heart is available, there is no data on the cellular location of opioid gene expression during development or in the adult. Using in situ hybridization and rat heart ranging from embryonic day 14 (E14) to adulthood, we have evaluated the distribution of gene expression for proenkephalin, proopiomelanocortin, and prodynorphin. With respect to preproenkephalin mRNA (PPE mRNA), message in the ventricle was abundant from E14 (the first time point examined) until shortly after birth, with a marked reduction noted on postnatal days 5, 10, and 21. Adults displayed considerable message, though less than in preparations of embryonic and neonatal heart. PPE mRNA was detected in epicardial, myocardial, and endocardial cells, as well as the walls of blood vessels, capillaries, and fibroblasts. Preproopiomelanocortin (POMC) mRNA was only found in adults, and was localized to the myocardium. Message for preprodynorphin could not be observed in the ventricles of developing or adult rats. These results are the first to define the temporal and spatial ontogeny of opioid gene expression with regard to the emergence of cardiac architecture. The data suggest that gene expression for proenkephalin is especially prevalent in embryonic and neonatal rats and may be related to the modulatory activity of the opioid growth factor, [Met5]-enkephalin, on cell proliferation and differentiation. The role of PPE and POMC mRNA in adult rat heart requires elucidation.     

Immunohistochemical localization of ORL-1 in the central nervous system of the rat

A novel member of the opioid receptor family (ORL-1) has been cloned from a variety of vertebrates. ORL-1 does not bind any of the classical opioids, although a high affinity endogenous agonist with close homology to dynorphin has recently been identified. We have generated a monoclonal antibody to the N-terminus of ORL-1 to map areas of receptor expression in rat central nervous system (CNS). Intense and specific immunolabeling was observed in multiple areas in the diencephalon, mesencephalon, pons/medulla, and spinal cord. In the telencephalon, intense labeling was observed in the neuropil throughout layers II-V in the neocortex, the anterior olfactory nuclear complex, the pyriform cortex, the CA1-CA4 fields and dentate gyrus of the hippocampus, and in many of the septal and basal forebrain areas. In contrast to other members of the opioid receptor family, light labeling for ORL-1 was observed in telencephalic areas such as caudate-putamen. In the cerebellum, ORL-1 immunoreactivity was only observed in the deep nuclei. Throughout the CNS the majority of labelling was localized to fiber processes and fine puncta, although labeled scattered perikarya were observed in a few brain areas such as the hilus dentate in the hippocampus and some nuclei in the brainstem and spinal cord. The present mapping study is consistent with the reported distribution of ORL-1 mRNA and provides the first immunohistochemical report on anatomical and cellular distribution of ORL-1 receptor in the rat CNS.     

Role of dynorphin and enkephalin in the regulation of striatal output pathways and behavior

Projection neurons in the striatum give rise to two output systems, the "direct" and "indirect" pathways, which antagonistically regulate basal ganglia output. While all striatal projection neurons utilize GABA as their principal neurotransmitter, they express different opioid peptide co-transmitters and also different dopamine receptor subtypes. Neurons of the direct pathway express the peptide dynorphin and the D1 dopamine receptor, whereas indirect pathway neurons express the peptide enkephalin and the D2 receptor. In the present review, we summarize our findings on the function of dynorphin and enkephalin in these striatal output pathways. In these studies, we used D1- or D2-receptor-mediated induction of immediate-early genes as a cellular response in direct or indirect projection neurons, respectively, to investigate the role of these opioid peptides. Our results suggest that the specific function of dynorphin and enkephalin is to dampen excessive activation of these neurons by dopamine and other neurotransmitters. Levels of these opioid peptides are elevated by repeated, excessive activation of these pathways, which appears to be an adaptive or compensatory response. Behavioral consequences of increased opioid peptide function in striatal output pathways may include behavioral sensitization (dynorphin) and recovery of motor function (enkephalin).     

Characterization of the aminopeptidase activity of epidermal leukotriene A4 hydrolase against the opioid dynorphin fragment 1-7

Leukotriene A4 hydrolase is a bifunctional cytosolic enzyme, which both hydrolyses leukotriene A4 (LTA4) into leukotriene B4 (LTB4) and exerts aminopeptidase activity against opioid peptides. In the present study we have investigated whether the peptides angiotensin I and II, bradykinin, kallidine, histamine, dynorphin fragment 1-7 and substance P can act as substrates for epidermal and neutrophil LTA4 hydrolase. Among the tested substrates, dynorphin fragment 1-7 was found to be the best substrate for the enzyme. The aminopeptidase activity of epidermal and neutrophil LTA4 hydrolase against dynorphin fragment 1-7 was further characterized. The enzyme was purified from human epidermis and human neutrophils by anion exchange chromatography (Q-Sepharose) and affinity chromatography on a column with the LTA4 hydrolase inhibitor bestatin coupled to AH-Sepharose. The incubation of the dynorphin fragment 1-7 with LTA4 hydrolase resulted in the formation of tyrosine. The presence of the N-terminal amino acid tyrosine is essential for the interaction of opioids with their receptors, and this finding indicates that the LTA4 hydrolase can inactivate dynorphin fragment 1-7. After the two purification steps no other aminopeptidases acting at the N-terminal tyrosine of dynorphin fragment 1-7 was present in the preparation. This was demonstrated by the abolishment of the degradation at the N-terminal end of dynorphin fragment 1-7 when preincubating the enzyme preparation with LTA4 before the incubation with the dynorphin fragment 1-7. The abolishment of the aminopeptidase activity shows that activation of the hydrolase part of the enzyme, with conversion of LTA4 into the potent proinflammatory compound LTB4, results in an inhibition of the aminopeptidase activity of the enzyme. As a result, the catabolism of dynorphin fragment 1-7 and probably of other opioid peptides is inhibited, resulting in sustained biological effects of these opioids. This phenomenon may be important for the maintenance of inflammation in skin conditions, such as psoriasis and atopic dermatitis, in which LTB4 is formed.     

Tyrosine hydroxylase-like immunoreactivity in the brain of the teleost fish Tinca tinca

The distribution of tyrosine hydroxylase-like immunoreactivity has been studied in the central nervous system of the tench (Tinca tinca) using a monoclonal antibody and the avidin-biotin-immunoperoxidase technique. Immunoreactive elements were found in all brain subdivisions. Thus, catecholaminergic neurons and fibers were detected in most nuclei of the ventral telencephalon and in the pars centralis and lateralis of the dorsal telencephalon. The diencephalon was the brain subdivision where largest density of immunoreactive elements were found, mainly located in the periventricular region. The mesencephalon and metencephalon only demonstrated immunoreactive fibers, and no immunoreactive cell bodies were observed in these regions. The myelencephalon showed three groups of immunoreactive neurons located at isthmal level, in the central medullary area, and at the medullary-spinal cord transition area. The distribution of catecholaminergic elements in the tench brain revealed a general pattern shared by most teleosts. The number and distribution of catecholaminergic elements was similar to those described in other teleostean species in the caudal region of the brain. However, noticeable differences were found in areas related to the integration of different sensory information, specially in the telencephalon and diencephalon, suggesting a relationship among the functional level of each sensorial system and the complexity of the catecholaminergic innervation of their integration regions. Additionally, this study revealed the presence of an important number of cerebrospinal fluid-contacting cells in the organum paraventricularis expressing tyrosine hydroxylase that in most investigated teleostean species were tyrosine hydroxylase-immunonegative despite they contained catecholamines. This data argues for distinct evolutionary patterns in the hypothalamic catecholaminergic system among different teleostean species.     

Age-related mu-, delta-and kappa-opioid ligands in respiratory-related brain regions of piglets: effect of prenatal cocaine

Neonates, as compared to older subjects, exhibit increased signs of relative respiratory suppression such as apnea, periodic breathing and only transient hyperventilatory response to hypoxia. Prenatal cocaine exposure exaggerates the respiratory pattern disturbances observed in infants. As endogenous opioids cause central suppression of breathing, we tested their possible involvement in these effects by assessing opioid content in respiratory-related brainstem regions of 2 to 5 (young) and 18 to 22 (older) day-old piglets, unexposed or preexposed to cocaine during 0.66 to 1.0 gestation. The selected ages represent distinct stages in the postnatal development of respiration. beta-Endorphin, methionine-enkephalin, dynorphin A and dynorphin B from the tractus solitarii, ambigualis, gigantoreticularis and parabrachialis medialis nuclei were separated by high performance liquid chromatography, then quantified by radioimmunoassays. Opioid content was higher in the brain regions of the young than of the older piglets, and increased after cocaine exposure in both age groups, but more in the young. These findings support the possible contribution of high opioid content to the relative suppression of respiratory function in early life, and to the exaggerated respiratory dysrhythmia observed in cocaine preexposed neonates.     

Expression of the conditioned NK cell activity is beta-endorphin dependent

We are interested in identifying the pathways which are responsible for triggering the conditioned enhancement of natural killer (NK) cell activity. Earlier studies have suggested that central opioid(s) are involved in eliciting the expression of the conditioned NK cell activity. The purpose of this study was to identify the central opioid peptides that allow the central nervous system (CNS) to communicate with the immune system. Mediators that activate the efferent pathway of communication between the CNS and immune system was examined by injection of the mediator via the cisterna magna (CM). Conditioning was used as a tool to show that the bi-directional communication between the CNS and the immune system does take place. We found that beta-endorphin but not dynorphin could stimulate NK cell activity, when beta-endorphin or dynorphin was injected into the CM. In addition, when anti-beta-endorphin or anti-dynorphin antibody was injected into the conditioned animals via CM the conditioned response was blocked by anti-beta-endorphin but not by anti-dynorphin antibody. These observations suggest that beta-endorphin appears to be one of the signals that is induced in the brain at the CS recall step of the conditioned response to trigger the elevation of NK cell activity.     

Effect of isolation conditions on brain regional enkephalin and !b-endorphin levels and vocalizations in 10-day-old rat pups.

Young rat pups were isolated from their dams under different conditions. The endogenous opioid peptides were measured in brain regions after isolation. Because there is no uptake mechanism for peptides released at the synapse and because released peptide is rapidly degraded enzymatically, decreases in peptide levels over this time course can be interpreted as release from terminals. No change was observed in either peptide in the hypothalamus, septum, or amygdala after isolation compared with controls. Significant decreases were seen in the midbrain after isolation. A comparison of peptide levels and ultrasonic vocalizations in the pups isolated in familiar, novel, or control conditions was also performed. Enkephalin levels in the midbrain were decreased in familiar and novel conditions, but in the brainstem opioid peptides were decreased only in the familiar condition. The greater involvement of the opioid peptides in the pups isolated in familiar conditions may contribute to the ability of naltrexone to block vocalization. (PsycINFO Database Record (c) 2010 APA, all rights reserved)