Prolyl-leucyl-glycinamide Research Articles

Evaluation of density functionals, SCC-DFTB, neglect of diatomic differential overlap (NDDO) models and molecular mechanics methods for prolyl-leucyl-glycinamide (PLG) and structural analogs

Prolyl-leucyl-glycinamide (PLG) is a unique endogenous peptide that modulates dopamine receptor subtypes of the D 2 receptor family within the CNS. We seek to elucidate the structural basis and molecular mechanism by which PLG and its analogs modulate dopamine receptors, toward the development of new therapeutics to treat Parkinson’s disease, tardive dyskinesia and schizophrenia. As a first step toward establishing a validated protocol for accurate computational modeling of PLG and associated peptidomimetic analogs, we evaluated the accuracy of density functional theory (DFT), wave function theory (WFT), and molecular mechanics (MM) calculations for PLG and for a library of structurally related small molecules. We first tested 12 local and nonlocal density functionals, Hartree–Fock (HF) theory, four “semiempirical” methods of the neglect of diatomic differential overlap (NDDO) type, and one self-consistent-charge nonorthogonal tight-binding (SCC-DFTB) method as implemented in two software suites, against coupled-cluster benchmark geometries for 4-methylthiazolidine, a small molecule that comprises key structural features present in our PLG-analog library. DFT and HF calculations were done with the MG3S augmented polarized triple-zeta basis set. We find that for 4-methylthiazolidine bond distances, DFT significantly outperforms NDDO, and both SCC-DFTB versions we evaluated perform worse than HF theory and are less accurate than 83% of the density functionals tested. The top five functionals for 4-methylthiazolidine were M05-2X, mPW1PW, B97-2, M06-2X, and PBEh, with mean unsigned errors (MUEs) in bond length of 0.0017, 0.0020, 0.0023, 0.0025 and 0.0027 Å, respectively. The widely used B3LYP functional ranked 11th out of 12 functionals evaluated, slightly below SCC-DFTB, and is significantly less accurate for 4-methylthiazolidine bond distances (MUE = 0.0095 Å) than the best local functional (M06-L, MUE = 0.0030 Å), which is far less computationally costly. Based on that initial analysis, we obtained new M05-2X benchmark geometric parameters for PLG and a library of 11 peptidomimetic derivatives, which we in turn used to examine the accuracy of thirty-four popular molecular mechanics (MM) force fields, four NDDO approaches, and SCC-DFTB for the full compound structures. Here, we found that ∼70% of the MM force fields tested superior to the best semiempirical and SCC-DFTB codings. Moreover, AMBER-type force fields proved most accurate among MM methods for this class of small-molecule peptidomimetics; the AMBER-type methods comprised eight out of the top 10 molecular mechanics options we tested.

Prolyl-leucyl-glycinamide shares some effects with oxytocin but decreases oxytocin levels

Oxytocin treatment in rats induces long-lasting antistress and growth promoting effects. This study investigated whether prolyl-leucyl-glycinamide (PLG) (the c-terminal tripeptide of oxytocin) or tocinoic acid (the ring structure of oxytocin) could induce some of these effects in male rats. For this purpose, PLG (2 or 10 mg/kg, s.c.) or tocinoic acid (1 mg/kg, s.c.) was administered to rats once a day for 3 or 5 days. Blood pressure, heart rate, spontaneous motor activity, nociceptive thresholds, and the survival of ischaemic musculocutaneous flaps were measured. In addition, endogenous oxytocin levels and plasma levels of some hormones known to be influenced by oxytocin were determined. PLG (2 mg/kg, s.c., but not 10 mg/kg, s.c.) decreased diastolic blood pressure ( p<0.05) and locomotor activity ( p<0.05). PLG (10 mg/kg, s.c.) decreased gastrin ( p<0.05) and endogenous oxytocin levels in plasma ( p<0.01). Tocinoic acid decreased locomotor activity ( p<0.05), but did not affect any of the other parameters measured. In conclusion, this study showed that both PLG and tocinoic acid decrease locomotor activity. In addition, PLG also induced some other effects similar to those induced by oxytocin treatment but when administered in high doses it decreased oxytocin levels.

PLGamide characterization and role in osmoregulation in leech brain

Neurons immunoreactive to an antiserum specifically directed against the Prolyl–Leucyl–Glycinamide peptide (PLGamide: Melanocyte Inhibiting Factor: MIF) were detected in the brain of the leech Theromyzon tessulatum. Radioimmunoassay titrations of the PLGamide-like material at different physiological stages of the life cycle indicated a maximal amount at stage 3B, which is correlated to phase of both maximal water uptake and coelomic vitellogenin accumulation. In vivo experiments demonstrate that this cerebral PLGamide-like material is an anti-diuretic factor that would act at stage 3B in order to permit a water uptake leading to water retention allowing coelomic yolk protein accumulation. In brains of the Gnatobdellid leech Hirudo medicinalis and the Pharyngobdellid leech Erpobdella octoculata, anti-PLGamide material was also detected with an amount not differing with the degree of sex maturation of the animals, confirming the link between osmoregulation and ovogenesis in rhynchobdellid leeches. Using a combination of biochemical techniques including high-pressure gel permeation chromatography followed by reversed-phase HPLC on brain extracts and Edman degradation, we demonstrated the presence of an authentic MIF-1 peptide in leech brain. Finally, since in vertebrates MIF-1 belongs to the non-classical opioid peptide family, we studied its binding displacement, in contrast to morphine, on μ-receptors and on nitric oxide (NO) release experiments in leech brain. PLGamide did not bind to μ-alkaloid opioid receptors and did not stimulate NO release.

Facilitation and attenuation of social recognition in rats by different oxytocin-related peptides

The memory-modulating effects of subcutaneously administered oxytocin and related peptides were investigated in the social recognition paradigm in rats. The attenuation of social recognition by 0.6 and 6 μg/kg of oxytocin was mimicked by the same doses of des-Gly 8-oxytocin, [pGlu 4,Cyt 6]oxytocin-(4-9) and prolyl-leucyl-glycinamide (PLG) and by 0.6 μg/kg of oxytocin-(1-6) and [pGlu 4,Cyt 6]oxytocin-(4-8). The peptide leucyl-glycinamide (LG) was not active in this respect. The facilitation of social recognition by 0.6 and 6 ng/kg of oxytocin was mimicked by the same doses of PLG and LG but not by the other peptides and the amino acid glycine. It is concluded that the active moieties for attenuation and facilitation of social memory reside in different parts of the oxytocin molecule, e.g. the 5–7 and 8–9 region, respectively.

Prolyl-leucyl-glycinamide, thyrotropin-releasing hormone and β-endorphin-(10–16), like antidepressants, antagonize melatonin-induced behavioural changes in rats

β-Endorphin-(10–16), as well as a variety of antidepressants, has been reported to block the behavioural changes induced by injecting melatonin into the nucleus accumbens. In the present study the influence of subcutaneously administered prolyl-leucyl-glycinamide (PLG) and thyrotropin-releasing hormone (TRH) on the behavioural changes induced by melatonin administration in the nucleus accumbens were investigated and compared with that of β-endorphin-(10–16). PLG and TRH were found to be as effective as β-endorphin-(10–16) in counteracting the melatonin-induced behavioural changes. The data suggest that these peptides may serve as a starting point for the development of a new class of antidepressants.

Effects of tizanidine on morphine physical dependence: Attenuation and intensification

It has previously been shown that subchronic and acute administration of L-asparaginase and glutaminase inhibitors D-Aspartic acid (D-ASP) and prolyl-leucyl-glycinamide (PLG) intensifies and attenuates morphine (M) physical dependence, respectively, by the inhibition of ASP and glutamic acid (GLU) production, and subsequently their normal releases. Tizanidine (TIZ) has long been known to be an α2-adrenoceptor agonist and inhibitor of ASP and GLU release. Therefore, in this study TIZ has been administered subchronically during the development of M physical dependence to rats in which M-containing pellets had been implanted or acurately 30 min before naloxone (NL)-induced abstinence syndrome. The subchronic administration of TIZ intensified NL-precipitated abstinence syndrome whereas its acute adminnistration attenuated it, as did D-ASP and PLG. On the other hand, TIZ added into the medium prevented the in vitro M-dependent-made guinea pig ileum from contracting following NL application. Furthermore, TIZ stopped the already started contraction by NL of the M-dependent ileum, which completely relaxed later. These effects of TIZ on M-dependent ileum were antagonized by the α2-adrenoceptor antagonist yohimbine. The intensification by subchronic TIZ administration of abstinence syndrome was attributed to the lesser release of ASP and GLU, which resulted in the larger blockade of M of ASPergic/GLUergic receptors due to the lesser release of their endogenous agonist ASP and GLU and consequently the higher upregulation of the receptors. The attenuation by acute TIZ administration of NL-precipitated abstinence syndrome was explained with lesser release of ASP and GLU and concomitantly the lesser stimulation of the receptors. The results of the in vitro experiments were considered supporting evidence for the fact that α2-adrenoceptor aginistic effect of causes the inhibition of ASP and GLU release and for this action TIZ has already been combined with dextromethorphan in the treatment of heroin addicts.

Effects of the oxytocin fragment prolyl-leucyl-glycinamide on sexual behavior in the rat

Prolyl-leucyl-glycinamide (PLG), a natural brain peptide, is identical in structure to the C-terminal of oxytocin. Moreover, PLG and oxytocin can act as opiate antagonists. Evidence that opiates and oxytocin have significant influences on reproductive behavior suggests that PLG may also be effective. Morphine and/or PLG were administered intraperitoneally to male and female rats and sexual behavior was observed. PLG (0.1–10 mg/kg) was found to facilitate female sexual behavior in Experiment 1. In Experiment 2, the ability of PLG to facilitate female receptivity was found to be progesterone dependent. In Experiment 3, tyrosine-prolyl-leucyl-glucinamide, a putative precursor to PLG, failed to facilitate lordosis. In Experiment 4, PLG failed to facilitate male sexual behavior. In Experiments 5 and 6, PLG did not affect morphine-induced inhibition of either male or female sexual behavior. These data suggest that PLG differentially affects female receptivity and male sexual behavior. The current results support the hypothesis that PLG is an active metabolite of oxytocin in the female, but do not provide evidence that PLG functions as an opiate antagonists of sexual behavior.

Intensification and attenuation of morphine dependence by D-aspartic acid and PLG

The inhibition by opiates and the sudden normalization by opioid antagonists of the brain L-asparaginase activity (BAA) have previously been reported to be the main factors in the development of physical dependence and the manifestation of precipitated abstinence syndrome, respectively. As a result, L-asparaginase inhibitors D-aspartic acid and prolyl-leucyl-glycinamide (PLG) were separately given to mice and rats either just after morphine (M)-containing pellet implantation or 15 min before naloxone (NL)-precipitated abstinence syndrome. The animals treated in this manner were used to assess the intensity of the physical dependence and to determine the BAA. D-ASP or PLG administration following pellet implantation significantly increased all of the observed signs such as flying, jumping, wet dog shake and writhing. When D-ASP or PLG were given 15 min before precipitated abstinence they significantly decreased the number of the signs. The determination of the BAA showed significant decreases or increases more or less parallel to the severity of the physical dependence on M. The intensification of physical dependence by D-ASP or PLG given just after the pellet implantation was attributed to their additional inhibitory effect to that of M on the BAA at the beginning of the physical dependence development. The attenuating effect of BAA inhibitors D-ASP or PLG administered before precipitated abstinence was explained with the prevention of the increase in the BAA.

Antiamnesic effects of D-pipecolic acid and analogues of Pro-Leu-Gly-NH 2 in rats

The antiamnesic effects of prolyl-leucyl-glycinamide (PLG) and analogues of this tripeptide were investigated in rats. Retrograde amnesia was induced by electroconvulsive shock treatment and the degree of amnesia was characterized by the attenuation of one-trial learning passive avoidance response. PLG resulted in dose-dependent attenuation of retrograde amnesia. Structural modifications included N-terminal protection, substitution of the C-terminal NH 2 group, replacement of the N-terminal amino acid, and replacement of the second amino acid of the tripeptide. Some tripeptides, all of them containing D-pipecolic acid instead of the N-terminal proline, were more effective than PLG. Therefore, D-pipecolic acid, D-pipecolamide and their N-terminally protected analogues were also investigated, and were found to have powerful antiamnesic effects.

Prolyl-leucyl-glycinamide (PLG): Inhibition of offensive aggression in mice

The effects on offensive aggression of the endogenous peptide-leucyl-glycinamide (PLG, MIF-1) and the exogenous opiate antagonist, naloxone, were examined in male mice. PLG (0.01–10 mg/Kg) reduced, in a dose-dependent manner, the incidence and intensity of offensive aggression in dominant resident mice. PLG was more potent than naloxone (1.0 mg/Kg). In a number of cases, PLG completely eliminated the display of offensive aggression towards intruder mice. These results raise the possibility that PLG may function as an “anti-aggressive” peptide whose actions may include antagonistic and/or modulatory influences on both opioid and non-opioid systems.

Neuropeptides and social behavior of rats tested in dyadic encounters

The effects of various neuropeptides on social behavior was studied in a test procedure in which 7-day isolated animals were tested together with non-isolated partners in dyadic encounters. The short-term isolation procedure increased the frequency and duration of social activities of the rats, but hardly affected non-social explorative behaviors of the animals. Systemic injection of certain neuropeptides, i.c. prolyl-leucyl-glycinamide (PLG), thyrotropin releasing hormone (TRH) and the ACTH 4–9 analog ORG 2766, reversed the isolation-induced increase in social activity, similarly as previcusly observed with antidepressant drugs. Subcutaneous treatment with β-endorphin, α-endorphin and des-Tyr-γ-endorphin increased social interaction in 7-day isolated animals. β-Endorphin enhanced social behavior of non-isolated rats as well, whereas γ-MSH decreased the social interactions of these animals. Both peptides affected especially social contact behavior. The potent action of β-endorphin suggests that this peptide and opioid systems may play a physiological role in social behavior. It is proposed that a possible functional antagonism between ACTH-like peptides, especially γ-MSH, and β-endorphin may operate in social behavior. The action of the peptides may be rather specific for social behavior, since none of the neuropeptides affected non-social explorative behaviors of the rats during the social interaction test.

Dose-dependent effects of prolyl-leucyl-glycinamide on morphine-induced analgesia, tolerance and dependence

Prolyl-leucyl-glycinamide (PLG) at a low dose (10 ng/mouse) administered intracerebroventricularly (i.c.v.) did not affect morphine analgesia, but produced a greater increase in the ED 50 of morphine-pretreated (100 mg/kg of morphine sulfate) mice as compared to control mice. PLG at doses of 10 and 100 μg/mouse antagonized morphine analgesia. Development of morphine tolerance was unaffected by 10 μg/mouse but antagonized by 100 μg/mouse of PLG. Development of morphine dependence was assessed by changes in body weight and temperature during naloxone-induced withdrawal. PLG (10 ng/mouse) potentiated, 10 μg/mouse had no effect and 100 μg/mouse antagonized development of morphine dependence. PLG at doses of 10 and 100 μg/mouse precipitated withdrawal in morphine-dependent mice. When mice were pretreated with 1.0 mg/kg naloxone i.p. 15 min before PLG, all doses of PLG had no effect on morphine analgesia, but potentiated the development of morphine tolerance and dependence. None of the doses of PLG altered whole brain levels of morphine. PLG did not alter the affinity of opioid receptors for etorphine or the maximal number of binding sites but PLG did exhibit a very weak affinity for opioid receptors. These results indicate that PLG potentiated development of morphine tolerance and dependence through a mechanism not involving opioid receptors. However, at very high doses it was a weak opioid receptor antagonist.

Effect of prolyl-leucyl-glycinamide and α-melanocyte-stimulating hormone on levorphanol-induced analgesia, tolerance and dependence

Prolyl-leucyl-glycinamide (PLG) at a low dose (10 ng/mouse) administered by an intracerebroventricular (i.c.v.) injection did not affect levorphanol analgesia, but PLG at higher doses (10 and 100 μg/mouse) and α-melanocyte-stimulating hormone (α-MSH) (10 ng/ mouse) antagonized levorphanol analgesia. Development of levorphanol tolerance was facilitated by 10 ng/mouse of PLG, unaffected by 10 μg/mouse of PLG, but antagonized by 100 μg/mouse of PLG and 10 ng/mouse of α-MSH. The effect of PLG on levorphanol dependence was assessed by changes in body weight and temperature during naloxone-induced withdrawal. PLG (10 ng/mouse) facilitated the development of levorphanol dependence, but 10 μg/mouse of PLG had no effect. PLG (100 μg/mouse) antagonized development of levorphanol dependence. PLG at doses of 10 and 100 μg/mouse precipitated withdrawal in levorphanol-dependent mice. α-MSH (10 ng/mouse) antagonized development of levorphanol dependence as evidenced by an increase in the ED50 of naloxone required to induce withdrawal jumping. These results indicate that PLG and α-MSH affected levorphanol-induced analgesia, tolerance and dependence in a qualitatively similar manner to their effect on morphine-induced analgesia, tolerance and dependence.

Neuropeptides related to neurohypophyseal hormones interfere with apomorphine-induced behavioral changes

The interaction between peptides related to neurohypophyseal hormones and brain dopaminergic systems was studied by investigating in rats the effect of these peptides on behavioral changes induced by graded doses of the specific dopamine agonist apomorphine. Low doses of this drug induce hypoactivity of the animals, while higher doses result in hyperactivity and stereotyped sniffing. Desglycinamide 9[Arg 8] vasopressin (DG-AVP), prolyl-leucyl-glycinamide (PLG) and oxytocin did not interfere with the behavioral responses induced by the higher doses of apomorphine. Peptide treatment made the rats more sensitive to apomorphine with respect to the drug induced hypoactivity. PLG and especially DG-AVP were more effective than oxytocin. It is concluded that these peptides may have a selective action on distinct dopaminergic receptor systems in the brain, that are presumably located presynaptically in the nucleus accumbens area.

Vasopressin release by D-aspartic acid, morphine and Prolyl-leucyl-glycinamide (PLG) in DI Brattleboro rats

The L-asparaginase activities of the brains of the Wistar, heterozygous and homozygous Brattleboro rats divided into three parts namely the anterior, middle and posterior which respectively contained cerebral cortex, hippocampus + midbrain + thalamus + hypothalamus and cerebellum + pons + medulla oblongata were estimated. The L-asparaginase activities of all the three parts in the homozygous Brattleboro rats were significantly higher than in the Wistar rats as well as in the heterozygous Brattleboro rats. Twenty min following the injections of 200 mg/kg D-aspartic acid, 20 mg/kg morphine, 200 mg/kg D-aspartic acid + 20 mg/kg morphine, 6 mg/kg prolyl-leucyl-glycinamide (PLG) and 6 mg/kg PLG + 20 mg/kg morphine the L-asparaginase activities of all three parts of the homozygous Brattleboro rat brains were found to be significantly inhibited. After having seen the suppressive effect of the drugs and their combinations used before the homozygous Brattleboro rats were given D-aspartic acid, morphine, D-aspartic acid + morphine, PLG and PLG + morphine for seven days. Then their plasma vasopressin levels were determined by RIA. The treatments applied to the homozygous Brattleboro rats caused the appearance of a significant amount vasopressin in the plasma. The results were interpreted as evidence for the fact that the inhibition of the brain L-asparaginase provides and/or accelerates the biosynthesis and/or release of vasopressin. As morphine has a vasopressin releasing and a brain L-asparaginase inhibiting effect the antidiuretic action of morphine was considered to be linked to its inhibitory effect on the brain L-asparaginase.

Arginine 8-vasopressin inhibits centrally induced pressor responses by involving hippocampal mechanisms

Administration of arginine 8-vasopressin (AVP) or prolyl-leucyl-glycinamide (PLG) into a lateral cerebral ventricle reduced the magnitude of systolic blood pressure increase (pressor response) induced by electrical stimulation of the mesencephalic reticular formation (MRF) in urethane-anesthetized rats. Bilateral destruction of the dorsal hippocampus prevented the action of AVP on the pressor response. However, the effect of PLG was only slightly reduced by hippocampal lesion. Microinjection of AVP in the dentate area of the dorsal hippocampus mimicked the action of intracerebroventricularly administered peptides. The effect of a single injection of AVP lasted at least for 60 min. Neither hippocampal damage nor peptide administrations resulted in changes in mean arterial blood pressure (basal BP). Bradycardiac response accompanied the BP increase during MRF stimulation. Hippocampal damage on intracerebroventricular administration of AVP and PLG failed to affect the cardiac response. Injection of AVP into the hippocampus tended to reduce the magnitude of cardiac responses caused by MRF stimulation. It is suggested that the inhibition by AVP of a pressor response produced by MRF stimulation involves the dorsal hippocampus. The action of PLG or related peptides seems to be, at least in part, through mechanisms not involving the hippocampus.

Feeding, drinking, urine osmolality in DI Brattleboro rats: Changes by morphine, naloxone, D-amino acids, prolyl-leucyl-glycinamide (PLG)

Bratteleboro rats placed in metabolism cages were injected with morphine (Mor), naloxone (Nal), D- and L-aspartic acid (D- and L-Asp), D-phenylalanine (D-Phe), D-leucine (D-Leu) and prolyl-leucyl-glycinamide (PLG), alone and in suitable combinations. Food and fluid intake, urine outflow, faeces weight, rectal temperature and urinary osmolality were determined at the end of seven hours period of time. Mor, Nal, D-Asp and PLG alone caused a significant decrease in food and fluid intake, urine volume and faeces weight and a significant increase in urinary osmolality being the osmolality of the Mor, D-Asp and PLG injected groups higher than 300 mOsmol/kg. The combination of Nal with Mor, D-Asp and PLG appeared to intensify the changes induced by Mor, D-Asp and PLG whereas L-Asp antagonized the majority of changes caused by Mor or PLG. The results were discussed in the light of the previous experimental findings.

Facilitation of morphine-induced tolerance and physical dependence by prolyl-leucyl-glycinamide

The pretreatment of mice with 30 mg/kg morphine s.c. did not alter the analgesic effect of morphine in mice pretreated with saline but decreased the analgesic effect of morphine in mice pretreated with prolyl-leucyl-glycin-amide (PLG). Tolerance was evaluated by the effect of PLG on morphine-induced enhancement of naloxone potency which is a measure of the capacity of naloxone to antagonize morphine-induced analgesia and is postulated to be an indicator of tolerance development. The naloxone potency of PLG-treated mice was 2-fold greater than that of control mice. PLG did not alter the whole brain levels of morphine, nor did it alter the naloxone potency in mice which were not pretreated with 30 mg/kg morphine. In mice treated with 100 mg/kg morphine or implanted with 50 mg morphine pellets for 24 or 72 h, the amount of naloxone required to induce jumping was not altered by PLG. However, PLG treatment did increase the hypothermia and body weight loss seen after naloxoneiinduced withdrawal. Administration of PLG to morphine-dependent mice 1 h prior to naloxone did not modify the resultant hypothermia or body weight loss. These results indicate that PLG facilitated the development of morphine tolerance and dependence.

Action of posterior pituitary neuropeptides on the nigrostriatal dopaminergic system

The effect of vasopressin, oxytocin and the C-terminal tripeptide of oxytocin, prolyl-leucyl-glycinamide (PLG), were tested on rotational behavior, following unilateral 6-OHDA-induced lesion of the dopaminergic cell bodies in the substantia nigra. Intraventricular injection of lysin 8-vasopressin, oxytocin or PLG caused ipsilateral (towards the lesioned side) rotation, as did peripheral administration of amphetamine. Direct local microinjection of the peptides into the substantia nigra on the intact side was without effect. The data suggest that posterior pituitary neuropeptides (vasopressin and oxytocin) caused presynaptic activation of the nigrostriatal dopaminergic terminals.

Modulation of substantia nigra self-stimulation by neuropeptides related to neurohypophyseal hormones

Neuropeptides related to hypothalamic-neurohypophyseal hormones affect brain functions. Among these are learning, consolidation and retrieval processes4, la. Pathways with neurohypophyseal hormone immunoreactivity have been described originating from hypothalamic nuclei with endings in various brain structures2, ~s, suggesting that these peptides may have direct effects on neuronal mechanisms in these structures. Behaviorally, a modulatory action of these peptides has been described using various test paradigms4, ~6, including acquisition of heroin self-administration 15. This particular behavior was attenuated by daily administration of desglycinamide 9, arginine s vasopressin (DG-AVP) and facilitated by that of prolyl-leucyl-glycinamide (PLG). It has been argued that the positively reinforcing characteristics of narcotics may be related to the action of these drugs on central reward structuresa, a. Thus, determination of the reinforcing efficacy of reward structures through studies of intracranial electrical self-stimulation (ICSS) behavior may provide information about the mechanisms mediating the reward induced by narcotics. The present study was performed to investigate the ability of DG-AVP and PLG to affect ICSS. The dopaminergic cell bodies in the substantia nigra-ventral tegmental area were selected as neuronal substrate for ICSS. Catecholaminergic, and in particular dopaminergic systems play a critical role in the rewarding quality of brain electrical stimulation 19 and of self-administered drugs 2°. Furthermore vasopressin and PLG affect catecholaminergic activity in various brain structures, including dopamine turnover in the nucleus caudatus 13,17. Details of the materials and methods used, are presented elsewhere 5. Briefly, 6 male Wistar rats were equipped with a twisted bipolar steel-wire electrode in the area of the substantia nigra (Pellegrino and Cushman 1° coordinates A 2.2, L 2.0, D 3.5.). After a recovery period of one week, the animals were shaped to self-stimulation in an operant conditioning chamber. Pressing the lever delivered an 0.5 sec biphasic squarewave train of impulses through the electrode on a continuous reinforcement schedule.