Kinetics of Formation and Asymmetrical Distribution of Hsp104-Bound Protein Aggregates in Yeast.

Budding yeast cells have a finite replicative life span; that is, a mother cell produces only a limited number of daughter cells before it slows division and dies. Despite the gradual aging of the mother cell, all daughters are born rejuvenated and enjoy a full replicative lifespan. It has been proposed that entry of mother cells into senescence is driven by the progressive accumulation and retention of damaged material, including protein aggregates. This additionally allows the daughter cells to be born damage free. However, the mechanism underlying such asymmetrical segregation of protein aggregates by mother and daughter cells remains controversial, in part because of the difficulties inherent in tracking the dynamics and fate of protein aggregates in vivo. To overcome such limitations, we have developed single-cell real-time imaging methodology to track the formation of heat-induced protein aggregates in otherwise unperturbed dividing cells. By combining the imaging data with a simple computational model of protein aggregation, we show that the establishment of asymmetrical partitioning of protein aggregates upon division is driven by the large bud-specific dilution rate associated with polarized growth and the absence of significant mother/bud exchange of protein aggregates during the budded phase of the cell cycle. To our knowledge, this study sheds new light on the mechanism of establishment of a segregation bias, which can be accounted for by simple physical arguments.

In vivo visualization of delta opioid receptors upon physiological activation uncovers a distinct internalization profile.

G-protein-coupled receptors (GPCRs) mediate numerous physiological functions and represent prime therapeutic targets. Receptor trafficking upon agonist stimulation is critical for GPCR function, but examining this process in vivo remains a true challenge. Using knock-in mice expressing functional fluorescent delta opioid receptors under the control of the endogenous promoter, we visualized in vivo internalization of this native GPCR upon physiological stimulation. We developed a paradigm in which animals were made dependent on morphine in a drug-paired context. When re-exposed to this context in a drug-free state, mice showed context-dependent withdrawal signs and activation of the hippocampus. Receptor internalization was transiently detected in a subset of CA1 neurons, uncovering regionally restricted opioid peptide release. Importantly, a pool of surface receptors always remained, which contrasts with the in vivo profile previously established for exogenous drug-induced internalization. Therefore, a distinct response is observed at the receptor level upon a physiological or pharmacological stimulation. Altogether, direct in vivo GPCR visualization enables mapping receptor stimulation promoted by a behavioral challenge and represents a powerful approach to study endogenous GPCR physiology.

Ligand-directed trafficking of the δ-opioid receptor in vivo: two paths toward analgesic tolerance.

δ-Opioid receptors are G-protein-coupled receptors that regulate nociceptive and emotional responses. It has been well established that distinct agonists acting at the same G-protein-coupled receptor can engage different signaling or regulatory responses. This concept, known as biased agonism, has important biological and therapeutic implications. Ligand-biased responses are well described in cellular models, however, demonstrating the physiological relevance of biased agonism in vivo remains a major challenge. The aim of this study was to investigate the long-term consequences of ligand-biased trafficking of the δ-opioid receptor, at both the cellular and behavioral level. We used δ agonists with similar binding and analgesic properties, but high [SNC80 ((+)-4-[(αR)-α-((2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl]-N,N-diethylbenzamide)]- or low [ARM390 (N,N-diethyl-4-(phenyl-piperidin-4-ylidenemethyl)-benzamide)]-internalization potencies. As we found previously, a single SNC80-but not ARM390-administration triggered acute desensitization of the analgesic response in mice. However, daily injections of either compound over 5 d produced full analgesic tolerance. SNC80-tolerant animals showed widespread receptor downregulation, and tolerance to analgesic, locomotor and anxiolytic effects of the agonist. Hence, internalization-dependent tolerance developed, as a result of generalized receptor degradation. In contrast, ARM390-tolerant mice showed intact receptor expression, but δ-opioid receptor coupling to Ca²+ channels was abolished in dorsal root ganglia. Concomitantly, tolerance developed for agonist-induced analgesia, but not locomotor or anxiolytic responses. Therefore, internalization-independent tolerance was produced by anatomically restricted adaptations leading to pain-specific tolerance. Hence, ligand-directed receptor trafficking of the δ-opioid receptor engages distinct adaptive responses, and this study reveals a novel aspect of biased agonism in vivo.

Lack of genotype effect on D1, D2 receptors and dopamine transporter binding in triple MOP-, DOP-, and KOP-opioid receptor knockout mice of three different genetic backgrounds.

We investigated D1, D2 receptors and dopamine transporter (DAT) binding levels in mice lacking all three opioid receptors and wild-type (WT) mice on three different genetic backgrounds. Quantitative autoradiography was used to determine the level of radioligand binding to the D1 and D2 receptors and DAT labeled with [(3)H]SCH23390, [(3)H]raclopride, and [(3)H]mazindol, respectively in triple-opioid receptor knockout (KO) and WT maintained on C57BL/6 (B6) and 129/SvEvTac (129) as well as C57BL/6 x 129/SvPas (B6 x 129) strains. No significant genotype effect was observed in D1, D2 receptors and DAT binding in any regions analyzed in any of the strains studied, suggesting that a lack of all three opioid receptors does not influence D1, D2 receptors and DAT expression, irrespective of their genetic strain background. However, strain differences were observed in D1 binding between the three strains of mice studied. Lower levels of D1 binding were observed in the substantia nigra of B6 x 129 WT mice compared with the 129 WT mice and in the olfactory tubercle of B6 x 129 WT compared with B6 WT and 129 WT mice. Lower levels of D1 binding were observed in the caudate putamen of B6 x 129 KO mice compared with 129 KO mice. In contrast, no significant strain differences were observed in D2 and DAT binding between the three strains of mice in any regions analyzed. Overall, these results indicate a lack of modulation of the dopaminergic system by the deletion of all three opioid receptors regardless of different background strains.

COSPLAY: An expandable toolbox for combinatorial and swift generation of expression plasmids in yeast.

A large number of genetic studies in yeast rely on the use of expression vectors. To facilitate the experimental approach of these studies, several collections of expression vectors have been generated (YXplac, pRS series, etc.). Subsequently, these collections have been expanded by adding more diversity to many of the plasmid features, including new selection markers and new promoter sequences. However, the ever growing number of plasmid features makes it unrealistic for research labs to maintain an up-to-date collection of plasmids. Here, we developed the COSPLAY toolbox: a Golden Gate approach based on the scheme of a simple modular plasmid that recapitulates and completes all the properties of the pRS plasmids. The COSPLAY toolbox contains a basal collection of individual functional modules. Moreover, we standardized a simple and rapid, software-assisted protocol which facilitates the addition of new personalized modules. Finally, our toolbox includes the possibility to select a genomic target location and to perform a single copy integration of the expression vector.

Excessive rDNA Transcription Drives the Disruption in Nuclear Homeostasis during Entry into Senescence in Budding Yeast.

Budding yeast cells undergo a limited number of divisions before they enter senescence and die. Despite recent mechanistic advances, whether and how molecular events are temporally and causally linked during the transition to senescence remain elusive. Here, using real-time observation of the accumulation of extrachromosomal rDNA circles (ERCs) in single cells, we provide evidence that ERCs build up rapidly with exponential kinetics well before any physiological decline. We then show that ERCs fuel a massive increase in ribosomal RNA (rRNA) levels in the nucleolus, which do not mature into functional ribosomes. This breakdown in nucleolar coordination is followed by a loss of nuclear homeostasis, thus defining a chronology of causally related events leading to cell death. A computational analysis supports a model in which a series of age-independent processes lead to an age-dependent increase in cell mortality, hence explaining the emergence of aging in budding yeast.

Inflammatory pain is enhanced in delta opioid receptor-knockout mice.

To examine the involvement of opioid receptors in inflammatory pain, we compared Complete Freund's Adjuvant-induced hyperalgesia in mice lacking mu, delta or kappa receptors under the same experimental conditions. Mechanical allodynia and thermal hyperalgesia were measured using von Frey filaments and the plantar test, respectively. All three receptor-knockout mice, as well as wild-type animals, developed inflammatory hyperalgesia following Complete Freund's Adjuvant administration. Mu-receptor mutants showed similar hyperalgesia to wild-types in the two tests. Kappa-receptor mutants exhibited enhanced mechanical allodynia compared with wild-type mice but similar thermal hyperalgesia. In contrast, mechanical allodynia and thermal hyperalgesia were both markedly augmented in delta-receptor mutants, indicating a role for an endogenous delta-receptor tone in the control of inflammatory pain. Treatment with the delta-selective agonist SNC80 produced antihyperalgesia, and this effect was abolished in the delta-receptor knockout mice. Altogether, these data demonstrate that delta receptors inhibit inflammatory pain when activated either endogenously or exogenously. We have previously shown enhanced neuropathic pain in delta-receptor knockout mice. The delta receptor definitely represents a promising target for treating chronic pain conditions.

Brain regional Fos expression elicited by the activation of mu- but not delta-opioid receptors of the ventral tegmental area: evidence for an implication of the ventral thalamus in opiate reward.

Both mu-opioid receptors (MORs) and delta-opioid receptors (DORs) are expressed in the ventral tegmental area (VTA) and are thought to be involved in the addictive properties of opiates. However, their respective contributions to opiate reward remain unclear. We used intracranial self-administration (ICSA) to study the rewarding effects of morphine microinjections into the VTA of male and female MOR-/- and DOR-/- mice. In brains of mice tested for intra-VTA morphine self-administration, we analyzed regional Fos protein expression to investigate the neural circuitry underlying this behavior. Male and female WT and DOR-/- mice exhibited similar self-administration performances, whereas knockout of the MOR gene abolished intra-VTA morphine self-administration at all doses tested. Naloxone (4 mg/kg) disrupted this behavior in WT and DOR mutants, without triggering physical signs of withdrawal. Morphine ICSA was associated with an increase in Fos within the nucleus accumbens, striatum, limbic cortices, amygdala, hippocampus, the lateral mammillary nucleus (LM), and the ventral posteromedial thalamus (VPM). This latter structure was found to express high levels of Fos exclusively in self-administering WT and DOR-/- mice. Abolition of morphine reward in MOR-/- mice was associated with a decrease in Fos-positive neurons in the mesocorticolimbic dopamine system, amygdala, hippocampus (CA1), LM, and a complete absence within the VPM. We conclude that (i) VTA MORs, but not DORs, are critical for morphine reward and (ii) the role of VTA-thalamic projections in opiate reward deserves to be further explored.

Morphine-induced analgesic tolerance, locomotor sensitization and physical dependence do not require modification of mu opioid receptor, cdk5 and adenylate cyclase activity.

Acute morphine administration produces analgesia and reward, but prolonged use may lead to analgesic tolerance in patients chronically treated for pain and to compulsive intake in opioid addicts. Moreover, long-term exposure may induce physical dependence, manifested as somatic withdrawal symptoms in the absence of the drug. We set up three behavioral paradigms to model these adaptations in mice, using distinct regimens of repeated morphine injections to induce either analgesic tolerance, locomotor sensitization or physical dependence. Interestingly, mice tolerant to analgesia were not sensitized to hyperlocomotion, whereas sensitized mice displayed some analgesic tolerance. We then examined candidate molecular modifications that could underlie the development of each behavioral adaptation. First, analgesic tolerance was not accompanied by mu opioid receptor desensitization in the periaqueductal gray. Second, cdk5 and p35 protein levels were unchanged in caudate-putamen, nucleus accumbens and prefrontal cortex of mice displaying locomotor sensitization. Finally, naloxone-precipitated morphine withdrawal did not enhance basal or forskolin-stimulated adenylate cyclase activity in nucleus accumbens, prefrontal cortex, amygdala, bed nucleus of stria terminalis or periaqueductal gray. Therefore, the expression of behavioral adaptations to chronic morphine treatment was not associated with the regulation of micro opioid receptor, cdk5 or adenylate cyclase activity in relevant brain areas. Although we cannot exclude that these modifications were not detected under our experimental conditions, another hypothesis is that alternative molecular mechanisms, yet to be discovered, underlie analgesic tolerance, locomotor sensitization and physical dependence induced by chronic morphine administration.

Controllable stress patterns over multi-generation timescale in microfluidic devices.

The generation of complex temporal stress patterns may be instrumental to investigate the adaptive properties of individual cells submitted to environmental stress on physiological timescale. However, it is difficult to accurately control stress concentration over time in bulk experiments. Here, we describe a microfluidics-based protocol to induce tightly controllable H2O2 stress in budding yeast while constantly monitoring cell growth with single cell resolution over multi-generation timescale. Moreover, we describe a simple methodology to produce ramping H2O2 stress to investigate the homeostatic properties of the H2O2 scavenging system.

Multiple inputs ensure yeast cell size homeostasis during cell cycle progression.

Coordination of cell growth with division is essential for proper cell function. In budding yeast, although some molecular mechanisms responsible for cell size control during G1 have been elucidated, the mechanism by which cell size homeostasis is established remains to be discovered. Here, we developed a new technique based on quantification of histone levels to monitor cell cycle progression in individual cells with unprecedented accuracy. Our analysis establishes the existence of a mechanism controlling bud size in G2/M that prevents premature onset of anaphase, and controls the overall size variability. While most G1 mutants do not display impaired size homeostasis, mutants in which cyclin B-Cdk regulation is altered display large size variability. Our study thus demonstrates that size homeostasis is not controlled by a G1-specific mechanism alone but is likely to be an emergent property resulting from the integration of several mechanisms that coordinate cell and bud growth with division.

Nonlinear feedback drives homeostatic plasticity in H2O2 stress response.

Homeostatic systems that rely on genetic regulatory networks are intrinsically limited by the transcriptional response time, which may restrict a cell's ability to adapt to unanticipated environmental challenges. To bypass this limitation, cells have evolved mechanisms whereby exposure to mild stress increases their resistance to subsequent threats. However, the mechanisms responsible for such adaptive homeostasis remain largely unknown. Here, we used live-cell imaging and microfluidics to investigate the adaptive response of budding yeast to temporally controlled H2O2 stress patterns. We demonstrate that acquisition of tolerance is a systems-level property resulting from nonlinearity of H2O2 scavenging by peroxiredoxins and our study reveals that this regulatory scheme induces a striking hormetic effect of extracellular H2O2 stress on replicative longevity. Our study thus provides a novel quantitative framework bridging the molecular architecture of a cellular homeostatic system to the emergence of nonintuitive adaptive properties.

Characterization of [3H] oxymorphone binding sites in mouse brain: Quantitative autoradiography in opioid receptor knockout mice.

Oxymorphone, one of oxycodone's metabolic products, is a potent opioid receptor agonist which is thought to contribute to the analgesic effect of its parent compound and may have high potential abuse liability. Nonetheless, the in vivo pharmacological binding profile of this drug is still unclear. This study uses mice lacking mu (MOP), kappa (KOP) or delta (DOP) opioid receptors as well as mice lacking all three opioid receptors to provide full characterisation of oxymorphone binding sites in the brain. Saturation binding studies using [3H]oxymorphone revealed high affinity binding sites in mouse brain displaying Kd of 1.7nM and Bmax of 147fmol/mg. Furthermore, we performed quantitative autoradiography binding studies using [3H]oxymorphone in mouse brain. The distribution of [3H]oxymorphone binding sites was found to be similar to the selective MOP agonist [3H]DAMGO in the mouse brain. [3H]Oxymorphone binding was completely abolished across the majority of the brain regions in mice lacking MOP as well as in mice lacking all three opioid receptors. DOP and KOP knockout mice retained [3H]oxymorphone binding sites suggesting oxymorphone may not target DOP or KOP. These results confirm that the MOP, and not the DOP or the KOP is the main high affinity binding target for oxymorphone.

Mu Opioid Receptors in Gamma-Aminobutyric Acidergic Forebrain Neurons Moderate Motivation for Heroin and Palatable Food.

BACKGROUND: Mu opioid receptors (MORs) are central to pain control, drug reward, and addictive behaviors, but underlying circuit mechanisms have been poorly explored by genetic approaches. Here we investigate the contribution of MORs expressed in gamma-aminobutyric acidergic forebrain neurons to major biological effects of opiates, and also challenge the canonical disinhibition model of opiate reward. METHODS: We used Dlx5/6-mediated recombination to create conditional Oprm1 mice in gamma-aminobutyric acidergic forebrain neurons. We characterized the genetic deletion by histology, electrophysiology, and microdialysis; probed neuronal activation by c-Fos immunohistochemistry and resting-state functional magnetic resonance imaging; and investigated main behavioral responses to opiates, including motivation to obtain heroin and palatable food. RESULTS: Mutant mice showed MOR transcript deletion mainly in the striatum. In the ventral tegmental area, local MOR activity was intact, and reduced activity was only observed at the level of striatonigral afferents. Heroin-induced neuronal activation was modified at both sites, and whole-brain functional networks were altered in live animals. Morphine analgesia was not altered, and neither was physical dependence to chronic morphine. In contrast, locomotor effects of heroin were abolished, and heroin-induced catalepsy was increased. Place preference to heroin was not modified, but remarkably, motivation to obtain heroin and palatable food was enhanced in operant self-administration procedures. CONCLUSIONS: Our study reveals dissociable MOR functions across mesocorticolimbic networks. Thus, beyond a well-established role in reward processing, operating at the level of local ventral tegmental area neurons, MORs also moderate motivation for appetitive stimuli within forebrain circuits that drive motivated behaviors.

Dissociation of analgesic and hormonal responses to forced swim stress using opioid receptor knockout mice.

Exposure to stress triggers hormonal and behavioral responses. It has been shown that the endogenous opioid system plays a role in some physiological reactions to stress. The opioid system was described to mediate analgesia induced by mild stressors and to modulate the activation of the hypothalamic-pituitary-adrenal axis. Our study assessed the contribution of opioid receptors in stress-induced analgesia and adrenocorticotropic hormone (ACTH) and corticosterone release by a genetic approach. We performed a parallel analysis of mice deficient in mu, delta, or kappa opioid receptors, as well as of triple opioid receptor knockout mice, following exposure to a mild stress (3-min swim at 32 degrees C). In wild-type mice, stress elicited an increase in jumping latency on the hot plate, which was influenced by gender and genetic background. This analgesic response was reversed both by naloxone and by the triple mutation, and decreased in mu and delta opioid receptor knockout females. In wild-type females, stress also delayed front- and hindpaw behaviors in the hot plate test and increased tail-flick latency in the tail immersion test. Opioid receptor deletion however did not affect these stress responses. In addition, stress produced an increase in ACTH and corticosterone plasma levels. This endocrine response remained unchanged in all mutant strains. Therefore our data indicate that, under our stress conditions, the endogenous opioid system is recruited to produce some analgesia whereas it does not influence hypothalamic-pituitary-adrenal axis activity. This implies that brain circuits mediating analgesic and hormonal responses to stress can be dissociated.

Delta opioid receptors expressed in forebrain GABAergic neurons are responsible for SNC80-induced seizures.

The delta opioid receptor (DOR) has raised much interest for the development of new therapeutic drugs, particularly to treat patients suffering from mood disorders and chronic pain. Unfortunately, the prototypal DOR agonist SNC80 induces mild epileptic seizures in rodents. Although recently developed agonists do not seem to show convulsant properties, mechanisms and neuronal circuits that support DOR-mediated epileptic seizures remain to be clarified. DORs are expressed throughout the nervous system. In this study we tested the hypothesis that SNC80-evoked seizures stem from DOR activity at the level of forebrain GABAergic transmission, whose inhibition is known to facilitate the development of epileptic seizures. We generated a conditional DOR knockout mouse line, targeting the receptor gene specifically in GABAergic neurons of the forebrain (Dlx-DOR). We measured effects of SNC80 (4.5, 9, 13.5 and 32 mg/kg), ARM390 (10, 30 and 60 mg/kg) or ADL5859 (30, 100 and 300 mg/kg) administration on electroencephalograms (EEGs) recorded in Dlx-DOR mice and their control littermates (Ctrl mice). SNC80 produced dose-dependent seizure events in Ctrl mice, but these effects were not detected in Dlx-DOR mice. As expected, ARM390 and ADL5859 did not trigger any detectable change in mice from both genotypes. These results demonstrate for the first time that SNC80-induced DOR activation induces epileptic seizures via direct inhibition of GABAergic forebrain neurons, and supports the notion of differential activities between first and second-generation DOR agonists.

A mu-delta opioid receptor brain atlas reveals neuronal co-occurrence in subcortical networks.

Opioid receptors are G protein-coupled receptors (GPCRs) that modulate brain function at all levels of neural integration, including autonomic, sensory, emotional and cognitive processing. Mu (MOR) and delta (DOR) opioid receptors functionally interact in vivo, but whether interactions occur at circuitry, cellular or molecular levels remains unsolved. To challenge the hypothesis of MOR/DOR heteromerization in the brain, we generated redMOR/greenDOR double knock-in mice and report dual receptor mapping throughout the nervous system. Data are organized as an interactive database offering an opioid receptor atlas with concomitant MOR/DOR visualization at subcellular resolution, accessible online. We also provide co-immunoprecipitation-based evidence for receptor heteromerization in these mice. In the forebrain, MOR and DOR are mainly detected in separate neurons, suggesting system-level interactions in high-order processing. In contrast, neuronal co-localization is detected in subcortical networks essential for survival involved in eating and sexual behaviors or perception and response to aversive stimuli. In addition, potential MOR/DOR intracellular interactions within the nociceptive pathway offer novel therapeutic perspectives.

A novel anxiogenic role for the delta opioid receptor expressed in GABAergic forebrain neurons.

BACKGROUND: The delta opioid receptor (DOR) is broadly expressed throughout the nervous system; it regulates chronic pain, emotional responses, motivation, and memory. Neural circuits underlying DOR activities have been poorly explored by genetic approaches. We used conditional mouse mutagenesis to elucidate receptor function in GABAergic neurons of the forebrain. METHODS: We characterized DOR distribution in the brain of Dlx5/6-CreXOprd1(fl/fl) (Dlx-DOR) mice and tested main central DOR functions through behavioral testing. RESULTS: The DOR proteins were strongly deleted in olfactory bulb and striatum and remained intact in cortex and basolateral amygdala. Olfactory perception, circadian activity, and despair-like behaviors were unchanged. In contrast, locomotor stimulant effects of SNC80 (DOR agonist) and SKF81297 (D1 agonist) were abolished and increased, respectively. The Dlx-DOR mice showed lower levels of anxiety in the elevated plus maze, opposing the known high anxiety in constitutive DOR knockout animals. Also, Dlx-DOR mice reached the food more rapidly in a novelty suppressed feeding task, despite their lower motivation for food reward observed in an operant paradigm. Finally, c-fos protein staining after novelty suppressed feeding was strongly reduced in amygdala, concordant with the low anxiety phenotype of Dlx-DOR mice. CONCLUSIONS: We demonstrate that DORs expressed in the forebrain mediate the described locomotor effect of SNC80 and inhibit D1-stimulated hyperactivity. Our data also reveal an unanticipated anxiogenic role for this particular DOR subpopulation, with a potential novel adaptive role. In emotional responses, DORs exert dual anxiolytic and anxiogenic roles, both of which may have implications in the area of anxiety disorders.

No evidence for G-protein-coupled epsilon receptor in the brain of triple opioid receptor knockout mouse.

Pharmacological approaches have defined the epsilon receptor as a beta-endorphin-preferring opioid receptor, described in rat vas deferens and in brain of several species. Only three opioid receptors-mu, delta and kappa-have been cloned and the existence of this additional subtype as a distinct protein remains controversial. Recently, the mouse brain epsilon receptor was detected in a G protein activation assay, as mediating residual beta-endorphin activity following pharmacological blockade of mu, delta and kappa receptors. To clarify whether this site is independent from mu, delta and kappa receptors, we performed beta-endorphin-induced [(35)S]GTPgammaS binding using mice lacking these three receptors (triple knockout mice). We tested both pons-medulla and whole brain preparations. beta-Endorphin strongly stimulated [(35)S]GTPgammaS binding in wild-type membranes but had no detectable effect in membranes from triple knockout mice. We conclude that the brain epsilon site involves mu, delta and/or kappa receptors, possibly coupled to nonclassical G proteins.

Knockout subtraction autoradiography: a novel ex vivo method to detect heteromers finds sparse KOP receptor/DOP receptor heterodimerization in the brain.

Several methodological approaches suggest that receptor heteromers exist in cell systems, but their presence in physiological tissue is widely contentious. We describe a novel method to determine if heterodimers exist in brain tissue sections using autoradiographic binding comparisons from single and double gene knockout mice, where tissues either have a full receptor complement and can form heterodimers, or are incapable of making heterodimers. We have tested this model, which we have named Knockout Subtraction Autoradiography, to determine if heterodimerisation of the kappa (KOP) and delta opioid (DOP) receptors occurs, as evidence from binding studies in cell systems suggest they are present in the brain. Using labeling of putative KOP receptor/DOP receptor heterodimers with either [(3)H]bremazocine or with [(3)H]naltrindole, two ligands which were used to provide evidence suggesting that these opioid receptor subtypes heterodimerize, we have applied a subtraction equation model based on the principle that receptor gene double knockout of either MOP receptor/KOP receptor (DOP receptor expression only) or MOP receptor/DOP receptor (KOP receptor expression only) produces tissue incapable of making the KOP receptor/DOP receptor heterodimer. We have shown in most brain regions that the labeling fits a simple additive model of monomer labeling, but that in a few brain regions opioid receptor heterodimerization does occur. The data does not support the conclusion that KOP receptor/DOP receptor heterodimerisation is widespread in the central nervous system, but does indicate that this novel methodology can detect heterodimerisation, when ligands with distinct binding affinities for monomer and heterodimer forms exist.