Comparison of Morphine and Endomorphin Analog ZH853 for Tolerance and Immunomodulation in a Rat Model of Neuropathic Pain.

µ-Opioid receptor agonists, the gold standard for analgesia, come with significant side effects when used chronically. Tolerance, defined as the decrease in analgesic activity after repeated use, remains a vital therapeutic obstacle as it increases the likelihood of dose escalation and potentially lethal side effects like respiratory depression. Previous experiments have indicated that the endomorphin-1 analog, ZH853, is a specific µ-opioid receptor agonist with reduced side effects like tolerance and glial activation following chronic central administration in pain-naïve animals. Here we investigated the effects of chronic, peripheral administration of µ-opioid receptor agonists following neuropathic injury. Though µ-opioids are effective at reducing neuropathic pain, they are not recommended for 1st line treatment due to negative side effects. Compared to chronic morphine, chronic ZH853 treatment led to decreased tolerance and reduced glial activation. Following twice daily intravenous injections, morphine was less potent and had a shorter duration of antinociception compared to ZH853. Chronic morphine, but not chronic ZH853, elevated markers of activation/inflammation of astrocytes (GFAP), microglia (Iba1), the proinflammatory cytokine TNFα and phosphorylated MAP kinase pp38. By contrast, chronic ZH853 reduced Iba1 and TNFα relative to both morphine and vehicle, suggesting anti-inflammatory properties with respect to these markers. GFAP and pp38 were not significantly different from vehicle but were significantly lower than morphine. This study demonstrates the effectiveness of chronic ZH853 for providing analgesia in a neuropathic pain state with reduced tolerance compared to morphine, potentially due to reductions in spinal glial activation. PERSPECTIVE: Neuropathic pain is generally undertreated, resistant to available medications, and opioid treatment is limited by side-effects. Here, we show that, compared to an equiantinociceptive dose of a stereotypical µ-opioid receptor agonist, morphine, chronic intravenous administration of endomorphin analog ZH853 led to prolonged antiallodynia, reduced tolerance, and inhibition of spinal cord neuroinflammation in male spared nerve-injured rats.

Endomorphin analog ZH853 shows low reward, tolerance, and affective-motivational signs of withdrawal, while inhibiting opioid withdrawal and seeking.

Currently available µ-opioid receptor agonist pharmacotherapies for opioid use disorder possess adverse effects limiting their use and, despite treatment, rates of relapse remain high. We previously showed that endomorphin analog ZH853 had no effect in rodent models that predict abuse liability in humans. Here we extended these findings by examining dependence liability and reinforcing properties in female rats and male rats with previous opioid exposure. The potential use of ZH853 in managing opioid use disorder was evaluated by examining its effect on opioid-seeking behavior and withdrawal. We found that ZH853 did not induce locomotor activation in male and female mice and was not self-administered by female rats. Relative to morphine, ZH853 led to similar somatic signs of withdrawal, but low affective-motivational signs of withdrawal, and absent changes in ventral tegmental area K(+)-Cl(-) co-transporter expression associated with reward dysregulation. The low abuse liability of ZH853 was further supported in oxycodone self-administering male rats, where ZH853 substitution extinguished opioid-seeking behavior. ZH853 priming also did not reinstate morphine conditioned place preference. Lastly, ZH853 inhibited oxycodone-seeking behavior during relapse after forced abstinence and decreased the expression of morphine withdrawal. These findings suggest the potential use of ZH853 as a safer opioid medication for long-term treatment of pain and opioid use disorder.

Discriminative Stimulus and Low Abuse Liability Effects of Novel Endomorphin Analogs Suggest a Potential Treatment Indication for Opioid Use Disorder.

Opioid dependence can be difficult to manage using existing pharmacotherapies. A long-acting opioid with low abuse liability that substitutes for a shorter-acting opioid may improve treatment of opioid use disorders (OUDs). We recently characterized an endomorphin (EM) analog (ZH853) that produced a longer duration of antinociception compared with morphine, but did not produce self-administration or several other adverse effects preclinically. Here, we further characterized ZH853 in tests of antinociception, abuse liability, and drug discrimination. A conditioned place preference (CPP) procedure, that included a locomotor activity assessment, was used to test abuse liability in rats. Subsequently, dopamine (DA) cell-somas located in the ventral tegmental area (VTA) from these rats were assessed by size using immunohistochemistry and Stereo Investigator software. A hot-plate antinociception test in male and female mice confirmed central penetration. Morphine-substitution effects of several EM analogs (ZH850, ZH831, and ZH853) were tested in a drug discrimination (DD) procedure in rats. Morphine produced dose-dependent CPP and locomotor sensitization and reduced the size of DA cell somas in VTA, whereas ZH853 did not produce any of these effects relative to control. The antinociceptive effects of ZH853 were µ-receptor selective since ß-funaltrexamine antagonized these effects. Rats responded on a morphine-trained lever when injected with ZH831 and ZH853 during DD experiments. The favorable morphine-substitution effects of these EM analogs relative to their low abuse liability indicate promising novel compounds that may improve treatment of OUD. SIGNIFICANCE STATEMENT: In this experiment, we investigated the preclinical effects of novel endomorphin analogs for use as substitution therapies for opioid use disorder, a problem that has contributed to an opioid overdose epidemic. Several endomorphin analogs substituted for morphine without producing adverse effects, including reward behaviors associated with abuse liability. These compounds have the potential to become important additional tools to treat opioid use disorders.

Morphine immunomodulation prolongs inflammatory and postoperative pain while the novel analgesic ZH853 accelerates recovery and protects against latent sensitization.

BACKGROUND: Numerous studies have identified the proinflammatory, pronociceptive effects of morphine which ultimately exacerbate pain. Our novel endomorphin analog ZH853 does not produce proinflammatory effects on its own and gives potent, long-lasting analgesia. This study investigates whether ZH853's lack of interaction with the neuroimmune system reduces the risk of prolonged pain. METHODS: Adult male Sprague-Dawley rats were subjected to one of two treatment paradigms. Either (1) chronic pain followed by chronic treatment with morphine, ZH853 or vehicle, or (2) chronic drug administered prior to pain induction. Complete Freund's adjuvant (CFA) was injected or paw incision surgery was performed on the left hind plantar foot pad. Drugs were administered through Alzet osmotic minipumps at a rate of 1 µl/h for 5 days at appropriate doses based on prior experiments. Animals were tested for mechanical allodynia and thermal hyperalgesia using von Frey filaments and the Hargreaves apparatus, respectively. Additionally, several gait parameters were measured using the CatWalk XT. When all animals had recovered from pain, 1 mg/kg of naltrexone was administered to test for development of latent sensitization (LS). A second set of animals was used to investigate dorsal horn inflammation following CFA and drug treatment. ANOVAs were used to assess differences between drug treatment groups. RESULTS: As expected, morphine increased and prolonged pain in all experiments compared to vehicle treatment. However, ZH853 treatment reduced the overall time spent in pain and the severity of pain scores compared to morphine. ZH853 not only reduced inflammation versus morphine treatment but also, in some instances, acted as an anti-inflammatory drug compared to vehicle treatment. Finally, ZH853 prevented the development of LS while vehicle- and morphine-treated animals showed robust relapse to pain. CONCLUSIONS: ZH853 has a favorable side effect profile versus morphine and provides superior analgesia in a number of pain states. We now know that chronic use of this compound reduces time spent in a chronic pain state, the opposite of common opioids like morphine, and reduces the risk of LS, making ZH853 an excellent candidate for clinical development in humans for inflammatory and postoperative pain.

Novel Endomorphin Analogs Are More Potent and Longer-Lasting Analgesics in Neuropathic, Inflammatory, Postoperative, and Visceral Pain Relative to Morphine.

Activation of the mu-opioid receptor provides the gold standard for pain relief, but most opioids used clinically have adverse effects that have contributed to an epidemic of overdose deaths. We recently characterized mu-opioid receptor selective endomorphin (EM) analogs that provide potent antinociception with reduction or absence of a number of side effects of traditionally prescribed opioids including abuse liability, respiratory depression, motor impairment, tolerance, and inflammation. The current study explores the effectiveness of these EM analogs relative to morphine in four major pain models by intrathecal as well as intravenous administration in male Sprague Dawley rats and subcutaneous administration in male CD-1 mice. In the spared nerve injury model of neuropathic pain, mechanical allodynia and mechanical hyperalgesia were assessed with von Frey and Randall-Selitto tests, respectively. In the paw incision model of postoperative pain, von Frey testing was used to assess mechanical allodynia and thermal hyperalgesia was evaluated with Hargreaves testing. In the Complete Freund's Adjuvant model of inflammatory pain, thermal hyperalgesia was assessed using Hargreaves testing. In CD-1 mice, visceral pain was assessed with the acetic acid writhing test. In all cases, EM analogs had equal or greater potency and longer duration of action relative to morphine. The data suggest that EM analogs, particularly analog 4 (ZH853), could provide effective therapy for a diverse spectrum of pain conditions with low risk of adverse side effects compared with currently used opioids such as morphine. PERSPECTIVE: Novel EM analogs show equal or greater potency and effectiveness relative to morphine in multiple pain models. Together with substantially reduced side effects, including abuse liability, the compounds show promise for addressing the critical need for effective pain relief as well as reducing the opioid overdose epidemic.

Endomorphin analog analgesics with reduced abuse liability, respiratory depression, motor impairment, tolerance, and glial activation relative to morphine.

Opioids acting at the mu opioid receptor (MOR) are the most effective analgesics, however adverse side effects severely limit their use. Of particular importance, abuse liability results in major medical, societal, and economic problems, respiratory depression is the cause of fatal overdoses, and tolerance complicates treatment and increases the risk of side effects. Motor and cognitive impairment are especially problematic for older adults. Despite the host of negative side effects, opioids such as morphine are commonly used for acute and chronic pain conditions. Separation of analgesia from unwanted effects has long been an unmet goal of opioid research. Novel MOR agonist structures may prove critical for greater success. Here we tested metabolically stable analogs of the endomorphins, endogenous opioids highly selective for the MOR. Compared to morphine, the analogs showed dramatically improved analgesia-to-side-effect ratios. At doses providing equal or greater antinociception than morphine in the rat, the analogs showed reduced a) respiratory depression, b) impairment of motor coordination, c) tolerance and hyperalgesia, d) glial p38/CGRP/P2X7 receptor signaling, and e) reward/abuse potential in both conditioned place preference and self-administration tests. Differential effects on glial activation indicate a mechanism for the relative lack of side effects by the analogs compared to morphine. The results suggest that endomorphin analogs described here could provide gold standard pain relief mediated by selective MOR activation, but with remarkably safer side effect profiles compared to opioids like morphine.

Pleiotropic opioid regulation of spinal endomorphin 2 release and its adaptations to opioid withdrawal are sexually dimorphic.

We studied adaptations to acute precipitated opioid withdrawal of spinal µ-opioid receptor (MOR)-coupled regulation of the release of endomorphin 2 (EM2). The release of this highly MOR-selective endogenous opioid from opioid-naive spinal tissue of male rats is subjected to MOR-coupled positive as well as negative modulation via cholera toxin-sensitive G(s) and pertussis toxin-sensitive G(i)/G(o), respectively. The net effect of this concomitant bidirectional modulation is inhibitory. MOR-coupled pleiotropic regulation of EM2 release is retained in opioid-withdrawn spinal tissue of male rats, but the balance of MOR-coupled inhibitory and facilitatory regulation shifted such that facilitatory regulation predominates. Augmented coupling of MOR to G(s) is causally associated with this change. Strikingly, pleiotropic characteristics of MOR-coupled regulation of spinal EM2 release and adaptations thereof to opioid withdrawal are male-specific. In females, MOR-coupled regulation of EM2 release from opioid-naive and -withdrawn spinal tissue does not have a significant G(s)-coupled facilitatory component, and MOR-coupled inhibition of EM2 release persists unabated in withdrawn preparations. The male-specific adaptations to chronic morphine that shift the relative predominance of opposing dual G protein-coupled MOR pathways provides a mechanism for mitigating inhibitory MOR signaling without losing MOR-coupled feedback regulation. These adaptations enable using endogenous EM2 as a substitute for morphine that had been precipitously removed. The sexually dimorphic functionality and regulation of spinal EM2/MOR-coupled signaling suggest the clinical utility of using sex-specific treatments for addiction that harness the activity of endogenous opioids.

A novel method to quantify histochemical changes throughout the mediolateral axis of the substantia gelatinosa after spared nerve injury: characterization with TRPV1 and substance P.

UNLABELLED: Nerve injury dramatically increases or decreases protein expression in the spinal cord dorsal horn. Whether the spatial distribution of these changes is restricted to the central innervation territories of injured nerves or could spread to adjacent territories in the dorsal horn is not understood. To address this question, we developed a simple computer software-assisted method to precisely distinguish and efficiently quantify immunohistochemical staining patterns across the mediolateral axis of the dorsal horn 2 weeks after transection of either the tibial and common peroneal nerves (thus sparing the sural branch, spared nerve injury, [SNI]), the tibial nerve, or the common peroneal and sural nerves. Using thiamine monophosphatase (TMP) histochemistry, we determined that central terminals of the tibial, common peroneal, sural, and posterior cutaneous nerves occupy the medial 35%, medial-central 20%, central-lateral 20%, and lateral 25% of the substantia gelatinosa, respectively. We then used these calculations to show that SNI reduced the expression of SP and TRPV1 immunoreactivity within the tibial and peroneal innervation territories in the L4 dorsal horn, without changing expression in the uninjured, sural sector. We conclude that SNI-induced loss of SP and TRPV1 in central terminals of dorsal horn is restricted to injured fibers. Our new method enables direct comparison of injured and uninjured terminals in the dorsal horn so as to better understand their relative contributions to mechanisms of chronic pain. PERSPECTIVE: A simple computer software-assisted algorithm was developed to precisely distinguish and efficiently quantify immunohistochemical staining patterns across the mediolateral axis of the dorsal horn after distal sciatic-branch transection. This method will facilitate a better understanding of the relative contribution of injured and uninjured terminals to mechanisms of chronic pain.

Opioidergic projections to sleep-active neurons in the ventrolateral preoptic nucleus.

Although opioids are known to influence sleep-wake regulation, the neuroanatomic substrate(s) mediating these effects remain unresolved. We hypothesized that the influence of opiates on sleep may be mediated, at least in part, by the ventrolateral preoptic nucleus (VLPO), a key cell group for producing behavioral sleep. By combining in situ hybridization for kappa and mu receptor mRNA with immunostaining of Fos expressed by VLPO cells during sleep we show that >85% of sleep-active VLPO neurons contain mRNA for either or both opioid receptors. Microinfusions of a kappa receptor agonist into the VLPO region increased NREM sleep by 51% during the subjective night, whereas a mu receptor agonist increased wakefulness by 60% during the subjective day. The sleep- and wake-promoting effects of the kappa and mu agonists were blocked by prior administration of their respective antagonist. Combining retrograde tracing from the VLPO with immunohistochemistry for dynorphin (Dyn, the endogenous kappa receptor agonist) or endomorphin 1 (EM1, the endogenous mu receptor agonist) we show that the central lateral parabrachial subnucleus (PBcl) provides Dyn inputs to the VLPO, whereas hypothalamic histaminergic neurons provide EM1 inputs to the VLPO. In summary, results from the present study suggest that central opioid inputs to the VLPO may play a role in sleep-wake regulation and that the VLPO likely mediates the hypnotic response to high levels of opioid analgesics.

Colocalization and shared distribution of endomorphins with substance P, calcitonin gene-related peptide, gamma-aminobutyric acid, and the mu opioid receptor.

The endomorphins are endogenous opioids with high affinity and selectivity for the mu opioid receptor (MOR, MOR-1, MOP). Endomorphin-1 (Tyr-Pro-Trp-Phe-NH(2); EM1) and endomorphin-2 (Tyr-Pro-Phe-Phe-NH(2); EM2) have been localized to many regions of the central nervous system (CNS), including those that regulate antinociception, autonomic function, and reward. Colocalization or shared distribution (overlap) of two neurotransmitters, or a transmitter and its cognate receptor, may imply an interaction of these elements in the regulation of functions mediated in that region. For example, previous evidence of colocalization of EM2 with substance P (SP), calcitonin gene-related peptide (CGRP), and MOR in primary afferent neurons suggested an interaction of these peptides in pain modulation. We therefore investigated the colocalization of EM1 and EM2 with SP, CGRP, and MOR in other areas of the CNS. EM2 was colocalized with SP and CGRP in the nucleus of the solitary tract (NTS) and with SP, CGRP and MOR in the parabrachial nucleus. Several areas in which EM1 and EM2 showed extensive shared distributions, but no detectable colocalization with other signaling molecules, are also described.

Reduced suppression of CO2-induced ventilatory stimulation by endomorphins relative to morphine.

Opioids are among the most effective analgesics, but a major limitation for their therapeutic usefulness is their induction of respiratory depression. Endomorphin-1 (EM1), in contrast to several other mu opioids, exhibits a threshold for respiratory depression that is well above its threshold for analgesia. Its effect on sensitivity to CO(2), however, remains unknown. Minute ventilation (V(E)) in 2, 4, and 6% CO(2) was measured before and after systemic administration of EM1, endomorphin-2 (EM2), DAMGO, and morphine in the conscious rat. EM1 and EM2 attenuated the hypercapnic ventilatory response (HCVR) only in high doses, while DAMGO and morphine diminished the HCVR in much lower doses. The ventilatory effects of high doses of all 4 agonists were blocked by the mu-opioid antagonist naloxone (0.4 mg/kg i.v.), but not by the peripherally restricted mu-opioid antagonist, methyl-naloxone (0.4 mg/kg i.v.). It was concluded that the endomorphins attenuated the HCVR only in large doses, well beyond the analgesic threshold, and did so through a centrally mediated mu-opioid mechanism.

Internalization and down-regulation of mu opioid receptors by endomorphins and morphine in SH-SY5Y human neuroblastoma cells.

The human neuroblastoma cell line, SH-SY5Y, was used to examine the effects of morphine and the endogenous opioid peptides, endomorphin-1 (EM-1) and endomorphin-2 (EM-2), on mu opioid receptor (MOR) internalization and down-regulation. Treatment for 24 h with EM-1, EM-2 or morphine at 100 nM, 1 microM and 10 microM resulted in a dose-dependent down-regulation of mu receptors. Exposure of cells to 10 microM EM-1 for 2.5, 5 and 24 h resulted in a time-dependent down-regulation of mu receptors. Down-regulation of mu receptors by morphine and EM-1 was blocked by treatment with hypertonic sucrose, consistent with an endocytosis-dependent mechanism. Sensitive cell-surface binding studies with a radiolabeled mu antagonist revealed that morphine was able to induce internalization of mu receptors naturally expressed in SH-SY5Y cells. EM-1 produced a more rapid internalization of mu receptors than morphine, but hypertonic sucrose blocked the internalization induced by each of these agonists. This study demonstrates that, like morphine, the endomorphins down-regulate mu opioid receptors in a dose- and time-dependent manner. This study also demonstrates that morphine, as well as EM-1, can induce rapid, endocytosis-dependent internalization of mu opioid receptors in SH-SY5Y cells. These results may help elucidate the ability of mu agonists to regulate the number and responsiveness of their receptors.

Endomorphins exit the brain by a saturable efflux system at the basolateral surface of cerebral endothelial cells.

Endomorphin-1 (EM-1) and endomorphin-2 (EM-2) are two highly selective mu-opiate receptor agonists. We recently demonstrated that EM-1 and EM-2 have a saturable transport system from brain-to-blood in vivo. Since the endothelial cells are the main component of the non-fenestrated microvessels of the blood-brain barrier (BBB), we examined whether these endogenous tetrapeptides have a saturable transport system in cultured cerebral endothelial cells. EM-1 and EM-2 binding and transport were studied in a transwell system in which primary mouse endothelial cells were co-cultured with rat glioma cells. We found that binding of both endomorphins was greater on the basolateral than the apical cell surface. Flux of EM-1 and EM-2 occurred predominantly in the basolateral to apical direction, each showing self-inhibition with an excess of the respective endomorphin. Transport was not influenced by the addition of the P-glycoprotein inhibitor, cyclosporin A. Neither the mu-opiate receptor agonist DAMGO nor the delta-opiate receptor agonist DPDPE had any effect on the transport. Thus, the results show that a saturable transport system for EM-1 and EM-2 occurs at the level of endothelial cells of the BBB, and unlike beta-endorphin and morphine, P-glycoprotein is not needed for the brain-to-blood transport. Cross-inhibition of the transport of each endomorphin by the other suggests a shared transport system that is different from mu- or delta-opiate receptors. As endormorphins are mainly produced in the CNS, the presence of the efflux system at the BBB could play an important role in pain modulation and neuroendocrine control.

Analgesic tolerance and cross-tolerance to i.c.v. endomorphin-1, endomorphin-2, and morphine in mice.

The present study examined the development of analgesic tolerance to endomorphin-1 (EM1), endomorphin-2 (EM2), and morphine, and cross-tolerance among these drugs. Male Swiss Webster mice were injected i.c.v. with EM1, EM2, morphine, or vehicle once daily for 5 days, and tested for analgesia in the tail flick test. To determine the extent of cross-tolerance, on the sixth day mice from each of the above groups received i.c.v. injections of EM1, EM2, morphine, or vehicle before analgesic testing. The development of tolerance to EM1 and EM2 closely resembled that of morphine. Complete, symmetrical cross-tolerance was observed between all drugs in the study. These results demonstrate a time-course and extent of tolerance similar to morphine, and support a common mechanism of action through the mu-opioid receptor.

Endomorphin-2 axon terminals contact mu-opioid receptor-containing dendrites in trigeminal dorsal horn.

The endomorphins represent a novel group of endogenous opioid peptides that have high affinity for the mu-opioid receptor (MOR1). Endomorphin-2 is present in high density in the spinal and trigeminal dorsal horns and is localized to primary afferents. If endomorphin-2 were an endogenous ligand for the MOR1, we would expect to find the receptor at cellular sites in close association with the peptide. We used dual-labeling immunocytochemical methods combined with electron microscopy to determine if a cellular substrate exists for functional interactions between endomorphin-2 and MOR1. We confirmed the localization of endomorphin-2 to unmyelinated axons and axon terminals in the trigeminal dorsal horn. A small proportion of these endomorphin-2 axons contained MOR1, but many of the dendritic targets of endomorphin-2 terminals contained MOR1. Consistent with previous studies, endomorphin-2 was contained primarily in dense core vesicles and MOR1 was located primarily at non-synaptic sites. These morphological characteristics are consistent with the hypothesis that peptides are released extra-synaptically and their receptors may be located at sites distal to the synaptic junction. These anatomical data support the hypothesis that endomorphin-2 is a ligand for MORs in the trigeminal dorsal horn, particularly at postsynaptic sites.

Morphological evidence of endomorphin as an agonist for the mu-opioid receptor in the rat spinal cord.

Endomorphin 2 is a newly discovered peptide that has high affinity and specificity for the mu-opioid receptor. One criterion for establishing that endomorphin serves as an endogenous agonist for the mu receptor is that it be anatomically distributed in close proximity to that receptor. We tested this idea with a preembedding double immunostaining technique to study synaptic relationships between them. The distributions of both endomorphin 2 and the mu-opioid receptor were similar in the dorsal horn of the cervical spinal cord at the light microscopic level. At the electron microscopic level, axon terminals with dense-cored vesicles containing endomorphin 2-like immunoreactivity were observed making mostly asymmetrical synapses on profiles immunostained for the mu-opioid receptor. The immunostaining for the mu-opioid receptor was found mostly in postsynaptic membranes in profiles having dendrite-like appearance. The results support the idea that endomorphin 2 is an endogenous ligand for the mu-opioid receptor. Furthermore, the results indicate that such a role is mediated at least in part through synaptic relationships.

Endomorphin-1 and -2 immunoreactive cells in the hypothalamus are labeled by fluoro-gold injections to the ventral tegmental area.

Endomorphin-1 and -2 (EM1, EM2) are endogenous opioids with high affinity and selectivity for the mu-opioid receptor. Cells expressing EM-like immunoreactivity (EM-LI) are present in the hypothalamus, and fibers containing EM-LI project to many brain regions, including the ventral tegmental area (VTA). The VTA is one of the most sensitive brain regions for the rewarding and locomotor effects of opioids. It contains mu-opioid receptors, which are thought to mediate gamma-aminobutyric acid-dependent disinhibition of dopamine transmission to the nucleus accumbens. We investigated whether hypothalamic EM-LI cells project to the VTA, where they could play a natural role in this circuitry. The retrograde tracer Fluoro-Gold (FG) was microinjected into the anterior or posterior VTA in rats. Nine days later, colchicine was injected, and 24 hours later, the animals were perfused and processed for fluorescence immunocytochemistry. Numerous FG-labeled cells were detected in the hypothalamus. Both EM1-LI and EM2-LI cells were present in the periventricular nucleus, between the dorsomedial and ventromedial hypothalamus and between the ventromedial and arcuate nuclei. Subpopulations of EM1-LI and EM2-LI cells were labeled by FG. Injections of FG to the anterior and posterior VTA were both effective in producing double-labeled cells, and an anterior-posterior topographical organization between the VTA and hypothalamus was observed. The results support the idea that some endomorphin-containing neurons in the hypothalamus project to the VTA, where they may modulate reward and locomotor circuitry.

Isolation and distribution of endomorphins in the central nervous system.

Endomorphin-1 (Tyr-Pro-Trp-Phe-NH2, EM-1) and endomorphin-2 (Tyr-Pro-Phe-Phe-NH2, EM-2) have the highest affinity and selectivity for the mu-opioid receptor (MOP-R) of all known mammalian opioids. They were isolated from bovine and human brain, and are structurally distinct from the other endogenous opioids. Both EM-1 and EM-2 have potent antinociceptive activity in a variety of animal models of acute, neuropathic and allodynic pain. They regulate cellular signaling processes in a manner consistent with MOP-R-mediated effects. The EMs are implicated in the natural modulation of pain by extensive data localizing EM-like immunoreactivity (EM-LI) near MOP-Rs in several regions of the nervous system known to regulate pain. These include the primary afferents and their terminals in the spinal cord dorsal horn, where EM-2 is well-positioned to modulate pain in its earliest stages of perception. In a nerve-injury model of chronic pain, a loss of spinal EM2-LI occurs concomitant with the onset of chronic pain. The distribution of the EMs in other areas of the nervous system is consistent with a role in the modulation of diverse functions, including autonomic, neuroendocrine and reward functions as well as modulation of responses to pain and stress. Unlike several other mu opioids, the threshold dose of EM-1 for analgesia is well below that for respiratory depression. In addition, rewarding effects of EM-1 can be separated from analgesic effects. These results indicate a favorable therapeutic profile of EM-1 relative to other mu opioids. Thus, the pharmacology and distribution of EMs provide new avenues both for therapeutic development and for understanding the neurobiology of opioids.

Morphological studies of the endomorphinergic neurons in the central nervous system.

Endomorphins (EMs) are newly found endogenous opioid peptides. Both endomorphin-1 (EM-1) and -2 (EM-2) are composed of four amino acids. Their high affinity and specificity for mu-opioid receptors have been confirmed by many physiological and pharmacological studies. In the present minireview, we discuss the distribution and localization of these peptides. While EM-2 is more prevalent in the spinal cord and lower brainstem, EM-1 is more widely and densely distributed throughout the brain than EM-2. We also discuss the possible coexistence of EM with other neurotransmitters. Finally, we introduce some new results regarding the ultrastructure and synaptic relationships of EM-2 obtained by the immunoelectron microscopic method.

Rewarding and psychomotor stimulant effects of endomorphin-1: anteroposterior differences within the ventral tegmental area and lack of effect in nucleus accumbens.

Endomorphin-1 (EM-1) is a recently isolated endogenous peptide having potent analgesic activity and high affinity and selectivity for the mu-opioid receptor. The present study was designed to investigate the rewarding and psychomotor stimulant effects of EM-1 in specific brain regions. We found that rats would learn without priming or response shaping to lever-press for microinjections of EM-1 into the ventral tegmental area (VTA); responding was most vigorous for high-dose injections into the posterior VTA. Rats did not learn to lever-press for microinjections of EM-1 into the nucleus accumbens (NAS) or regions just dorsal to the VTA. Lever-pressing for EM-1 in the VTA was extinguished when vehicle was substituted for the peptide and was reinstated when EM-1 reinforcement was re-established. Conditioned place preference was established by EM-1 injections into the posterior but not the anterior VTA or the NAS. Injection of EM-1 (0.1-1.0 nmol) into the posterior VTA induced robust increases in locomotor activity, whereas injections into the anterior VTA had very weak locomotor-stimulating effects. When injected into the NAS, EM-1 (0.1-10.0 nmol) did not affect locomotor activity. The present findings implicate the posterior VTA as a highly specific and sensitive site for opioid reward and suggest a role for EM-1-containing projections to the posterior VTA in the rewarding effects of other reinforcers.