Biomedical subjects

John E Pintar

Publications and source records attributed to John E Pintar.

At least 19 recordsLinked to original sources

PubMed2006

Differential involvement of endogenous opioids in sucrose consumption and food reinforcement.

Endogenous opioids within the central nervous system are postulated to mediate hedonic aspects of feeding behavior. To identify the relevant endogenous opioid receptor ligands, mice lacking one or two of the opioid peptide families beta-endorphin, enkephalins or dynorphins were tested for sucrose preference in a two-bottle free-choice drinking paradigm under drug-naïve conditions and following treatment with an opioid antagonist (1 mg/kg naloxone i.p.) or saline. Basal sucrose consumption was unaltered in all of the knockout genotypes compared to their congenic wild-type C57BL/6 littermates during 0.5 and 6 h access to a bottle containing 2, 4, 8, or 16% sucrose and a second bottle containing water. Moreover, all mutant genotypes and wildtype mice exhibited a similar compensatory decrease in overnight food intake following the extra caloric load from 6 h sucrose access. Although these basal responses to sucrose were unaffected by the knockout genotypes, naloxone reduced sucrose consumption by 50% compared to saline treatment during the first 0.5 h in wild-type and beta-endorphin knockout mice, but had no effect in enkephalin knockouts, beta-endorphin and enkephalin double knockouts, or dynorphin knockouts. These data suggest that naloxone reduces sucrose consumption in wild-type mice by blocking endogenous enkephalin and/or dynorphin signaling, but not beta-endorphin. Dynorphin knockouts in the current study had bar-pressing responses for a palatable food reinforcer in an operant procedure under free-feeding conditions similar to wild-type mice while we found in a previous study that beta-endorphin and enkephalin knockout mice had reduced motivation to respond [Hayward MD, Pintar JE, Low MJ. Selective reward deficit in mice lacking beta-endorphin and enkephalin. J Neurosci 2002;22:8251-8258.]. We conclude from these studies directly comparing three strains of opioid peptide knockout mice that enkephalin and dynorphin can modulate sucrose preference but are not necessary to support sucrose consumption. However, dynorphin was not necessary to support wildtype levels of operant responding suggesting that only enkephalin and beta-endorphin modulate conditioned food reinforcement.

Animals↗

PubMed2006

Demonstration of lysosomal localization for the mammalian ependymin-related protein using classical approaches combined with a novel density shift method.

Most newly synthesized soluble lysosomal proteins are delivered to the lysosome via the mannose 6-phosphate (Man-6-P)-targeting pathway. The presence of the Man-6-P post-translational modification allows these proteins to be affinity-purified on immobilized Man-6-P receptors. This approach has formed the basis for a number of proteomic studies that identified multiple as yet uncharacterized Man-6-P glycoproteins that may represent new lysosomal proteins. Although the presence of Man-6-P is suggestive of lysosomal function, the subcellular localization of such candidates requires experimental verification. Here, we have investigated one such candidate, ependymin-related protein (EPDR). EPDR is a protein of unknown function with some sequence similarity to ependymin, a fish protein thought to play a role in memory consolidation and learning. Using classical subcellular fractionation on rat brain, EPDR co-distributes with lysosomal proteins, but there is significant overlap between lysosomal and mitochondrial markers. For more definitive localization, we have developed a novel approach based upon a selective buoyant density shift of the brain lysosomes in a mutant mouse lacking NPC2, a lysosomal protein involved in lipid transport. EPDR, in parallel with lysosomal markers, shows this density shift in gradient centrifugation experiments comparing mutant and wild type mice. This approach, combined with morphological analyses, demonstrates that EPDR resides in the lysosome. In addition, the lipidosis-induced density shift approach represents a valuable tool for identification and validation of both luminal and membrane lysosomal proteins that should be applicable to high throughput proteomic studies.

Animals↗

PubMed2006

The role of prohormone convertase-2 in hypothalamic neuropeptide processing: a quantitative neuropeptidomic study.

Prohormone convertase (PC) 1/3 and 2 are involved in the generation of neuropeptides from their precursors. A quantitative peptidomic approach was used to explore the role PC2 plays in the processing of hypothalamic peptides. In this approach, extracts from mice lacking PC2 activity and from wild-type littermates were labeled with isotopic tags, combined, fractionated on a reverse phase HPLC column, and analyzed by electrospray ionization mass spectrometry. Altogether, 53 neuropeptides or other peptides derived from secretory pathway proteins were identified and sequenced using tandem mass spectrometry. These peptides arise from 21 distinct proteins: proenkephalin, proopiomelanocortin, prodynorphin, protachykinin A and B, procholecystokinin, promelanin-concentrating hormone, proneurotensin, proneuropeptide Y, provasopressin, pronociceptin/orphanin, prothyrotropin-releasing hormone, cocaine- and amphetamine-regulated transcript, chromogranin A and B, secretogranin II, prohormone convertase 1 and 2, propeptidyl-amidating monooxygenase, and proteins designated proSAAS and VGF. Approximately one third of the peptides found in wild-type mice were not detectable in PC2 knock-out mice, and another third were present at levels ranging from 25 to 75% of wild-type levels. Comparison of the cleavage sites suggests that sequences with a Trp, Tyr and/or Pro in the P1' or P2' position, or a basic residue in the P3 position, are preferentially cleaved by PC2 and not by other enzymes present in the secretory pathway.

Amino Acid Sequence↗

PubMed2006

Diminished growth and enhanced glucose metabolism in triple knockout mice containing mutations of insulin-like growth factor binding protein-3, -4, and -5.

IGF-I and IGF-II are essential regulators of mammalian growth, development and metabolism, whose actions are modified by six high-affinity IGF binding proteins (IGFBPs). New lines of knockout (KO) mice lacking either IGFBP-3, -4, or -5 had no apparent deficiencies in growth or metabolism beyond a modest growth impairment (approximately 85-90% of wild type) when IGFBP-4 was eliminated. To continue to address the roles of these proteins in whole animal physiology, we generated combinational IGFBP KO mice. Mice homozygous for targeted defects in IGFBP-3, -4, and -5 remain viable and at birth were the same size as IGFBP-4 KO mice. Unlike IGFBP-4 KO mice, however, the triple KO mice became significantly smaller by adulthood (78% wild type) and had significant reductions in fat pad accumulation (P < 0.05), circulating levels of total IGF-I (45% of wild type; P < 0.05) and IGF-I bioactivity (37% of wild type; P < 0.05). Metabolically, triple KO mice showed normal insulin tolerance, but a 37% expansion (P < 0.05) of beta-cell number and significantly increased insulin secretion after glucose challenge, which leads to enhanced glucose disposal. Finally, triple KO mice demonstrated a tissue-specific decline in activation of the Erk signaling pathway as well as weight of the quadriceps muscle. Taken together, these data provide direct evidence for combinatorial effects of IGFBP-3, -4, and -5 in both metabolism and at least some soft tissues and strongly suggest overlapping roles for IGFBP-3 and -5 in maintaining IGF-I-mediated postnatal growth in mice.

Adipose Tissue↗

PubMed2006

Antinociceptive and hypothermic effects of Salvinorin A are abolished in a novel strain of kappa-opioid receptor-1 knockout mice.

Salvia divinorum is a natural occurring hallucinogen that is traditionally used by the Mazatec Indians of central Mexico. The diterpene salvinorin A was identified as an active component of S. divinorum over 20 years ago, but only recently has biochemical screening indicated that a molecular target of salvinorin A in vitro is the kappa-opioid receptor. We have examined whether salvinorin A, the C2-substituted derivative salvinorinyl-2-propionate, and salvinorin B can act as kappa-opioid receptor agonists in vivo. We found that following intracerebroventricular injection over a dose range of 1 to 30 microg of both salvinorin A and salvinorinyl-2-propionate produces antinociception in wild-type mice but not in a novel strain of kappa-opioid receptor knockout mice. Moreover, both salvinorin A and salvinorinyl-2-propionate reduce rectal body temperature, similar to conventional kappa-opioid receptor agonists, in a genotype-dependent manner. In addition, we determined that salvinorin A has high affinity for kappa 1- but not kappa 2-opioid receptors, demonstrating selectivity for this receptor subclass. Finally, treatment over the same dose range with salvinorin B, which is inactive in vitro, produced neither antinociceptive nor hypothermic effects in wild-type mice. These data demonstrate that salvinorin A is the active component of S. divinorum, selective for kappa(1)-opioid receptors, and that salvinorin A and specific structurally related analogs produce behavioral effects that require the kappa-opioid receptor.

Analgesics↗

PubMed2006

Prior activation of kappa opioid receptors by U50,488 mimics repeated forced swim stress to potentiate cocaine place preference conditioning.

Repeated forced-swim stress (FSS) produced analgesia, immobility and potentiation of cocaine-conditioned place preference (CPP) in wild-type C57Bl/6 mice, but not in littermates lacking the kappa opioid receptor (KOR) gene. These results were surprising because kappa agonists are known to produce conditioned place aversion and to suppress cocaine-CPP when coadministered with cocaine. The possibility that disruption of the kappa system blocked the stress response by adversely affecting the hypothalamic-pituitary axis was examined by measuring plasma corticosterone levels. However, disruption of the dynorphin/kappa system by gene deletion or receptor antagonism did not reduce the FSS-induced elevation of plasma corticosterone levels. A second explanation for the difference is that kappa receptor activation caused by FSS occurred prior to cocaine conditioning rather than contemporaneously. To test this hypothesis, we measured the effects of the kappa agonist (trans)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl]benzeneacetamide (U50,488) administered to mice at various intervals preceding cocaine conditioning. The results showed that the interaction between the kappa system and cocaine reinforcement depended on the timing of the drug pairing. Mice given U50,488 60 min prior to cocaine showed a robust, nor-BNI-sensitive potentiation of cocaine-CPP, whereas administration 15 min before cocaine significantly suppressed cocaine-CPP. In the absence of cocaine, U50,488 given 60 min prior to saline conditioning produced no place preference, whereas administration 15 min before saline conditioning produced significant place aversion. The results of this study suggest that kappa receptor activation induced by FSS prior to the cocaine-conditioning session may be both necessary and sufficient for potentiation of the reinforcing actions of cocaine.

3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cycloh↗

PubMed2005

Deletion of peptide amidation enzymatic activity leads to edema and embryonic lethality in the mouse.

Peptidylglycine alpha-amidating monooxygenase (PAM) catalyzes the COOH-terminal amidation of peptide hormones. We previously had found high expression of PAM in several regions of the developing rodent. To determine the function of PAM during mouse embryogenesis, we produced a null mutant of the PAM gene. Homozygous mutants die in utero between e14.5 and e15.5 with severe edema that is likely due to cardiovascular deficits. These defects include thinning of the aorta and carotid arteries and are very similar to those of the recently characterized adrenomedullin (AM) gene KO despite the presence of elevated immunoreactive AM in PAM KO embryos. No peptide amidation activity was detected in PAM mutant embryos, and there was no moderation of the AM-like phenotype that could be expected if any alternative peptide amidation mechanism exists in the mouse. Despite the proposed contribution of amidated peptides to neuronal cell proliferation, no alteration in neuroblast proliferation was observed in homozygous mutant embryos prior to lethality. Mice heterozygous for the mutant PAM allele develop normally and express wildtype levels of several amidated peptides despite having one half the wildtype levels of PAM activity and PAM protein. Nonetheless, both an increase in adiposity and a mild glucose intolerance developed in aged (>10 months) heterozygous mice compared to littermate controls. Ablation of PAM thus demonstrates an essential function for this gene during mouse development, while alterations in PAM activity in the adult may underlie more subtle physiologic effects.

Adrenomedullin↗

PubMed2005

Endogenous kappa-opioid receptor systems regulate mesoaccumbal dopamine dynamics and vulnerability to cocaine.

Genetic and pharmacological approaches were used to examine kappa-opioid receptor (KOR-1) regulation of dopamine (DA) dynamics in the nucleus accumbens and vulnerability to cocaine. Microdialysis revealed that basal DA release and DA extraction fraction (Ed), an indirect measure of DA uptake, are enhanced in KOR-1 knock-out mice. Analysis of DA uptake revealed a decreased Km but unchanged Vmax in knock-outs. Knock-out mice exhibited an augmented locomotor response to cocaine, which did not differ from that of wild-types administered a behavioral sensitizing cocaine treatment. The ability of cocaine to increase DA was enhanced in knock-outs, whereas c-fos induction was decreased. Although repeated cocaine administration to wild types produced behavioral sensitization, knock-outs exhibited no additional enhancement of behavior. Administration of the long-acting KOR antagonist nor-binaltorphimine to wild-type mice increased DA dynamics. However, the effects varied with the duration of KOR-1 blockade. Basal DA release was increased whereas Ed was unaltered after 1 h blockade. After 24 h, release and Ed were increased. The behavioral and neurochemical effects of cocaine were enhanced at both time points. These data demonstrate the existence of an endogenous KOR-1 system that tonically inhibits mesoaccumbal DA neurotransmission. Its loss induces neuroadaptations characteristic of "cocaine-sensitized" animals, indicating a critical role of KOR-1 in attenuating responsiveness to cocaine. The increased DA uptake after pharmacological inactivation or gene deletion highlights the plasticity of mesoaccumbal DA neurons and suggests that loss of KOR-1 and the resultant disinhibition of DA neurons trigger short- and long-term DA transporter adaptations that maintain normal DA levels, despite enhanced release.

Analysis of Variance↗

PubMed2005

Strain-dependent influences on the hypothalamo-pituitary-adrenal axis profoundly affect the 7B2 and PC2 null phenotypes.

Two null mouse models have previously been created to study the role of the prohormone convertase (PC2) and its helper protein 7B2; unexpectedly, the phenotypes of these two nulls differ profoundly, with the 7B2 but not the PC2 null dying at 5 wk. The genetic backgrounds of these two models differ, with the 7B2 null in a 129/SvEv (129) background and the PC2 null in a mixed C57BL/N6:129/SvEv (B6:129) background. Because background can contribute greatly to phenotype, we have here examined strain influence on the hypothalamo-pituitary-adrenal (HPA) axis and glucose levels in wild-type, 7B2 null, and PC2 null mice. Wild-type B6 and 129 mice differed in basal corticosterone and glucose levels. When 7B2 nulls were transferred onto the B6 background, they survived and showed greatly decreased circulating corticosterone and increased blood glucose levels, most likely due to the comparatively higher adrenal resistance of the B6 strain to ACTH stimulation. Circulating ACTH levels were increased over wild-type in the B6 7B2 null but did not reach levels as high as the 129 7B2 null. Conversely, when the mixed-strain PC2 nulls were bred into the 129 background at the N6 generation, they began to exhibit the Cushing's-like phenotype characteristic of 129 7B2 null mice and died before 6 wk of age. Taken together, these results indicate that background effects are critical because they increase the phenotypic differences between the 7B2 and PC2 nulls and play a life-or-death role in the ACTH hypersecretion syndrome present in both 129 nulls.

Adrenocorticotropic Hormone↗

PubMed2005

Unresponsiveness of mu-opioid receptor knockout mice to lipopolysaccharide-induced fever.

Recently, we demonstrated that lipopolysaccharide (LPS)-induced fever could be suppressed by a selective mu-opioid receptor antagonist, indicating that the mu-opioid system is involved in the LPS fever. In the present study, to confirm the role of the mu-opioid system in the pathogenesis of LPS fever, we used mice lacking the mu-opioid receptor. In the wild type (WT), following intraperitoneal (i.p.) injection of 100 microg kg(-1) of LPS, body temperature (T(b)) increased approximately 1 degrees C and remained elevated during the 360-min recording period. In the mu-opioid receptor knockout (MOR-KO) mice, the administration of 100 microg kg(-1) i.p. of LPS did not induce fever during the recording period. Saline by itself, given i.p., did not alter the T(b), either in WT or MOR-KO. These results confirm that the mu-opioid system is involved in LPS-induced fever.

Animals↗

PubMed2005

Decreased firing frequency of midbrain dopamine neurons in mice lacking mu opioid receptors.

Dopamine neurons originating in the midbrain and projecting to cortico-limbic and motor structures are one of the major neuronal substrates implicated in the reinforcing properties of drugs of abuse. The output of this system is largely determined by its impulse activity (amount and pattern of firing activity). Several intrinsic and synaptic factors can influence dopamine neuronal activity and, consequently, addiction liability. Pharmacological studies indicate that mu-opioid receptors and their activation by endogenous opioids may play an important role. In the present study, we use a genetic approach to better understand the role of mu-opioid receptors in modulating dopamine neuronal activity in vivo. Using in vivo extracellular single-unit recordings, we show that mice lacking mu-opioid receptors exhibit lower firing rates of dopamine neurons compared with their wild-type littermates. Although we observed no overall changes in bursting activity compared with wild-type mice, animals lacking mu-opioid receptors exhibited a higher proportion of regular-spiking cells that lacked bursting activity. These findings are the first to emphasize the critical role of mu-opioid receptors in modulating action potential output of dopamine neurons in vivo using a genetic approach. They also provide a possible underlying mechanism for the decreased reinforcing properties of drugs of abuse that was previously observed in mice lacking mu-opioid receptors.

Action Potentials↗

PubMed2005

Embryonic gene expression and pro-protein processing of proSAAS during rodent development.

In vitro assays have demonstrated that peptides derived from the recently-identified proSAAS precursor inhibit prohormone convertase 1 (PC1) suggesting that this novel peptide may function as an endogenous inhibitor of PC1. To further understand the role of proSAAS in vivo, we have investigated the expression of proSAAS mRNA and processing of proSAAS during pre- and early postnatal rodent development. In situ hybridization showed that, by embryonic day 12.5 (e12.5) in the rat, proSAAS mRNA was present in essentially all differentiating neurons in the mantle layer of the myelencephalon, metencephalon, diencephalon, spinal cord and several sympathetic ganglia. During later stages of prenatal development, widespread proSAAS expression continues in post-mitotic neurons of both the CNS and PNS and begins in endocrine cells of the anterior and intermediate pituitary. Although proSAAS expression overlaps with PC1 in several regions, its overall expression pattern is significantly more extensive, suggesting that proSAAS may be multifunctional during development. Processed forms of proSAAS are present by at least mid-gestation with marked accumulation of two C-terminal forms, comprising the PC1 inhibitory fragment of proSAAS.

Animals↗

PubMed2004

Neuropathic pain activates the endogenous kappa opioid system in mouse spinal cord and induces opioid receptor tolerance.

Release of endogenous dynorphin opioids within the spinal cord after partial sciatic nerve ligation (pSNL) is known to contribute to the neuropathic pain processes. Using a phosphoselective antibody [kappa opioid receptor (KOR-P)] able to detect the serine 369 phosphorylated form of the KOR, we determined possible sites of dynorphin action within the spinal cord after pSNL. KOR-P immunoreactivity (IR) was markedly increased in the L4-L5 spinal dorsal horn of wild-type C57BL/6 mice (7-21 d) after lesion, but not in mice pretreated with the KOR antagonist nor-binaltorphimine (norBNI). In addition, knock-out mice lacking prodynorphin, KOR, or G-protein receptor kinase 3 (GRK3) did not show significant increases in KOR-P IR after pSNL. KOR-P IR was colocalized in both GABAergic neurons and GFAP-positive astrocytes in both ipsilateral and contralateral spinal dorsal horn. Consistent with sustained opioid release, KOR knock-out mice developed significantly increased tactile allodynia and thermal hyperalgesia in both the early (first week) and late (third week) interval after lesion. Similarly, mice pretreated with norBNI showed enhanced hyperalgesia and allodynia during the 3 weeks after pSNL. Because sustained activation of opioid receptors might induce tolerance, we measured the antinociceptive effect of the kappa agonist U50,488 using radiant heat applied to the ipsilateral hindpaw, and we found that agonist potency was significantly decreased 7 d after pSNL. In contrast, neither prodynorphin nor GRK3 knock-out mice showed U50,488 tolerance after pSNL. These findings suggest that pSNL induced a sustained release of endogenous prodynorphin-derived opioid peptides that activated an anti-nociceptive KOR system in mouse spinal cord. Thus, endogenous dynorphin had both pronociceptive and antinociceptive actions after nerve injury and induced GRK3-mediated opioid tolerance.

Animals↗

PubMed2004

A role for heterodimerization of mu and delta opiate receptors in enhancing morphine analgesia.

Opiates such as morphine are the choice analgesic in the treatment of chronic pain. However their long-term use is limited because of the development of tolerance and dependence. Due to its importance in therapy, different strategies have been considered for making opiates such as morphine more effective, while curbing its liability to be abused. One such strategy has been to use a combination of drugs to improve the effectiveness of morphine. In particular, delta opioid receptor ligands have been useful in enhancing morphine's potency. The underlying molecular basis for these observations is not understood. We propose the modulation of receptor function by physical association between mu and delta opioid receptors as a potential mechanism. In support of this hypothesis, we show that mu-delta interacting complexes exist in live cells and native membranes and that the occupancy of delta receptors (by antagonists) is sufficient to enhance mu opioid receptor binding and signaling activity. Furthermore, delta receptor antagonists enhance morphine-mediated intrathecal analgesia. Thus, heterodimeric associations between mu-delta opioid receptors can be used as a model for the development of novel combination therapies for the treatment of chronic pain and other pathologies.

Analgesia↗

PubMed2004

The delta-opioid receptor participates in T-cell development by promoting negative selection.

The delta-opioid receptor-1 (DOR-1) as well as delta-opioid enkephalin peptides are expressed during maturation of T cells, although the functional significance of their expression remains unclear. Based on results which show that the administration of the highly selective delta-opioid agonist D-Pen2, D-Pen5]enkephalin (DPDPE) induces an altered pattern of T-cell differentiation in fetal thymic organ culture (FTOC), we hypothesized that DOR-1 is involved in the negative selection process. Our results show that superantigen-induced clonal deletion is promoted by DPDPE and significantly impaired in DOR-1-deficient mice. These results suggest that delta-opioids may play a homeostatic role in the negative selection process during T-cell development.

Analgesics, Opioid↗

PubMed2004

Operant self-administration of ethanol in C57BL/6 mice lacking beta-endorphin and enkephalin.

To test whether endogenous opioid peptides are necessary for the rewarding effects of ethanol, we examined operant oral self-administration of ethanol in mice congenic to the C57BL/6J strain but lacking expression of beta-endorphin, enkephalin or both peptides. The influences of prandial state, schedule interval and type, and ethanol concentration were all examined. Food-restricted subjects were tested in postprandial and preprandial states and subsequently at normal body weight when feeding ad libitum (ad lib). Operant studies were conducted using fixed ratio (FR) intervals of 2 and 8 as well as a progressive ratio (PR) interval of 2. The main significant effect relevant to our hypothesis was increased responding by female mice lacking beta-endorphin under ad lib feeding conditions and only for lower ethanol concentrations (3% and 6%). Importantly, all subjects including those lacking both beta-endorphin and enkephalins learned to self-administer ethanol similarly to wild-type mice and maintained responding for ethanol under a variety of procedural variables. Consequently, the two opioid peptides believed to be the endogenous ligands for the micro-opioid receptor (MOR) were not necessary to shape or perpetuate ethanol-reinforced operant responding. These results suggest that the rewarding effects of ethanol do not require beta-endorphin or enkephalin signaling.

Alcohol Drinking↗

PubMed2004

Gene discovery by microarray: identification of novel genes induced during growth factor-mediated muscle cell survival and differentiation.

Peptide growth factors regulate cell fate by activating distinct signal transduction pathways that ultimately influence gene expression. Insulin-like growth factors (IGFs) play central roles in controlling somatic growth and participate in skeletal muscle development and regeneration. In cultured muscle cells, IGF action is critical both for maintaining viability during the transition from proliferating to differentiating myoblasts and for facilitating differentiation. By contrast, platelet-derived growth factor (PDGF) can sustain cell survival but inhibits differentiation. Here we examine the genetic programs that accompany IGF and PDGF action in myoblasts. Through analysis of high-density oligonucleotide arrays containing approximately 36,000 mouse probe sets, we identify 90 transcripts differentially induced by IGF-I, including 28 muscle-specific genes and 33 previously unannotated mRNAs, and 55 transcripts specifically stimulated by PDGF, including 14 unknowns. Detailed study of one IGF-induced mRNA shows that it encodes a protein related to a recently characterized repulsive guidance molecule postulated to regulate neuronal targeting during development. Our results demonstrate the power of transcriptional profiling for gene discovery and provide opportunities for investigating new proteins potentially involved in different aspects of growth factor action in muscle.

Animals↗

PubMed2004

Mu-opioid receptor knockout mice show diminished food-anticipatory activity.

We have previously suggested that during or prior to activation of anticipatory behaviour to a coming reward, mu-opioid receptors are activated. To test this hypothesis schedule induced food-anticipatory activity in mu-opioid receptor knockout mice was measured using running wheels. We hypothesized that mu-knockout mice show little food-anticipatory activity. In wildtype mice we observed that food-anticipatory activity increased proportional to reduced food intake levels during daily scheduled food access, and thus reflects the animal's physiological need for food. mu-Knockout mice do not adjust their schedule induced running wheel behaviour prior to and during feeding time in the same way as wildtype mice; rather than showing more running wheel activity before than during feeding, they showed an equal amount of activity before and during feeding. As food-anticipatory activity is dependent on the mesolimbic dopamine system and mu-opioid receptors regulate dopaminergic activity, these data suggest a change in the dopamine system's activity in mu-knockout mice. As we observed that mu-knockout mice tended to show a stronger locomotor activity response than wildtype mice to the indirect dopamine agonist d-amphetamine, it appears that the dopaminergic system per se is intact and sensitive to activation. We found no differences in the expression of pro-opiomelanocortin, a precursor of endogenous endorphin, in the arcuate nucleus between mu-knockout mice and wildtype mice during restricted feeding, showing that the mu-opioid receptor does not regulate endogenous endorphin levels. These data overall suggest a role for mu-opioid receptors in adapting reward related behaviour to the requirements of the environment.

Amphetamine↗