IL-1R-Mediated Activation of Renal Sensory Nerves in DOCA-Salt Hypertension.

BACKGROUND: Clinical trials of renal denervation for the treatment of hypertension have shown a variety of off-target improvements in conditions associated with sympathetic overactivity. This may be due to the ablation of sympathoexcitatory afferent renal nerves, which are overactive under conditions of renal inflammation. Renal IL (interleukin)-1ß is elevated in the deoxycorticosterone acetate-salt model of hypertension, and its activity may be responsible for the elevation in afferent renal nerve activity and arterial pressure. METHODS: Continuous blood pressure recording of deoxycorticosterone acetate-salt mice with IL-1R (IL-1 receptor) knockout or antagonism was used individually and combined with afferent renal denervation (ARDN) to assess mechanistic overlap. Protein quantification and histological analysis of kidneys were performed to characterize renal inflammation. RESULTS: ARDN attenuated deoxycorticosterone acetate-salt hypertension (-20±2-Δmm Hg mean arterial pressure [MAP] relative to control at study end) to a similar degree as total renal denervation (-21±2-Δmm Hg MAP), IL-1R knockout (-16±4-Δmm Hg MAP), or IL-1R antagonism (-20±3-Δmm Hg MAP). The combination of ARDN with knockout (-18±2-Δmm Hg MAP) or antagonism (-19±4-Δmm Hg MAP) did not attenuate hypertension any further than ARDN alone. IL-1R antagonism was found to have an acute depressor effect (-15±3-Δmm Hg MAP, day 10) in animals with intact renal nerves but not those with ARDN. CONCLUSIONS: These findings suggest that IL-1R signaling is partially responsible for the elevated afferent renal nerve activity, which stimulates central sympathetic outflow to drive deoxycorticosterone acetate-salt hypertension.

AAV-mediated gene transfer to colon-innervating primary afferent neurons.

Investigation of neural circuits underlying visceral pain is hampered by the difficulty in achieving selective manipulations of individual circuit components. In this study, we adapted a dual AAV approach, used for projection-specific transgene expression in the CNS, to explore the potential for targeted delivery of transgenes to primary afferent neurons innervating visceral organs. Focusing on the extrinsic sensory innervation of the mouse colon, we first characterized the extent of dual transduction following intrathecal delivery of one AAV9 vector and intracolonic delivery of a second AAV9 vector. We found that if the two AAV9 vectors were delivered one week apart, dorsal root ganglion (DRG) neuron transduction by the second vector was greatly diminished. Following delivery of the two viruses on the same day, we observed colocalization of the transgenes in DRG neurons, indicating dual transduction. Next, we delivered intrathecally an AAV9 vector encoding the inhibitory chemogenetic actuator hM4D(Gi) in a Cre-recombinase dependent manner, and on the same day injected an AAV9 vector carrying Cre-recombinase in the colon. DRG expression of hM4D(Gi) was demonstrated at the mRNA and protein level. However, we were unable to demonstrate selective inhibition of visceral nociception following hM4D(Gi) activation. Taken together, these results establish a foundation for development of strategies for targeted transduction of primary afferent neurons for neuromodulation of peripheral neural circuits.

Periglomerular afferent innervation of the mouse renal cortex.

Recent studies using a novel method for targeted ablation of afferent renal nerves have demonstrated their importance in the development and maintenance of some animal models of hypertension. However, relatively little is known about the anatomy of renal afferent nerves distal to the renal pelvis. Here, we investigated the anatomical relationship between renal glomeruli and afferent axons identified based on transient receptor potential vanilloid 1 channel (TRPV1) lineage or calcitonin gene related peptide (CGRP) immunolabeling. Analysis of over 6,000 (10,000 was accurate prior to the removal of the TH data during the review process) glomeruli from wildtype C57BL/6J mice and transgenic mice expressing tdTomato in TRPV1 lineage cells indicated that approximately half of all glomeruli sampled were closely apposed to tdTomato+ or CGRP+ afferent axons. Glomeruli were categorized as superficial, midcortical, or juxtamedullary based on their depth within the cortex. Juxtamedullary glomeruli were more likely to be closely apposed by afferent axon subtypes than more superficial glomeruli. High-resolution imaging of thick, cleared renal slices and subsequent distance transformations revealed that CGRP+ axons closely apposed to glomeruli were often found within 2 microns of nephrin+ labeling of glomerular podocytes. Furthermore, imaging of thick slices suggested that CGRP+ axon bundles can closely appose multiple glomeruli that share the same interlobular artery. Based on their expression of CGRP or tdTomato, prevalence near glomeruli, proximity to glomerular structures, and close apposition to multiple glomeruli within a module, we hypothesize that periglomerular afferent axons may function as mechanoreceptors monitoring glomerular pressure. These anatomical findings highlight the importance of further studies investigating the physiological role of periglomerular afferent axons in neural control of renal function in health and disease.

Long-term reversal of chronic pain behavior in rodents through elevation of spinal agmatine.

Chronic pain remains a significant burden worldwide, and treatments are often limited by safety or efficacy. The decarboxylated form of L-arginine, agmatine, antagonizes N-methyl-d-aspartate receptors, inhibits nitric oxide synthase, and reverses behavioral neuroplasticity. We hypothesized that expressing the proposed synthetic enzyme for agmatine in the sensory pathway could reduce chronic pain without motor deficits. Intrathecal delivery of an adeno-associated viral (AAV) vector carrying the gene for arginine decarboxylase (ADC) prevented the development of chronic neuropathic pain as induced by spared nerve injury in mice and rats and persistently reversed established hypersensitivity 266 days post-injury. Spinal long-term potentiation was inhibited by both exogenous agmatine and AAV-human ADC (hADC) vector pre-treatment but was enhanced in rats treated with anti-agmatine immunoneutralizing antibodies. These data suggest that endogenous agmatine modulates the neuroplasticity associated with chronic pain. Development of approaches to access this inhibitory control of neuroplasticity associated with chronic pain may yield important non-opioid pain-relieving options.

Adeno-associated virus-mediated gene transfer of arginine decarboxylase to the central nervous system prevents opioid analgesic tolerance.

Agmatine, a decarboxylated form of L-arginine, prevents opioid analgesic tolerance, dependence, and self-administration when given by both central and systemic routes of administration. Endogenous agmatine has been previously detected in the central nervous system. The presence of a biochemical pathway for agmatine synthesis offers the opportunity for site-specific overexpression of the presumptive synthetic enzyme for local therapeutic effects. In the present study, we evaluated the development of opioid analgesic tolerance in ICR-CD1 mice pre-treated with either vehicle control or intrathecally delivered adeno-associated viral vectors (AAV) carrying the gene for human arginine decarboxylase (hADC). Vehicle-treated or AAV-hADC-treated mice were each further divided into two groups which received repeated delivery over three days of either saline or systemically-delivered morphine intended to induce opioid analgesic tolerance. Morphine analgesic dose-response curves were constructed in all subjects on day four using the warm water tail flick assay as the dependent measure. We observed that pre-treatment with AAV-hADC prevented the development of analgesic tolerance to morphine. Peripheral and central nervous system tissues were collected and analyzed for presence of hADC mRNA. In a similar experiment, AAV-hADC pre-treatment prevented the development of analgesic tolerance to a high dose of the opioid neuropeptide endomorphin-2. Intrathecal delivery of anti-agmatine IgG (but not normal IgG) reversed the inhibition of endomorphin-2 analgesic tolerance in AAV-hADC-treated mice. To summarize, we report here the effects of AAV-mediated gene transfer of human ADC (hADC) in models of opioid-induced analgesic tolerance. This study suggests that gene therapy may contribute to reducing opioid analgesic tolerance.

Targeting the somatosensory system with AAV9 and AAV2retro viral vectors.

Adeno-associated viral (AAV) vectors allow for site-specific and time-dependent genetic manipulation of neurons. However, for successful implementation of AAV vectors, major consideration must be given to the selection of viral serotype and route of delivery for efficient gene transfer into the cell type being investigated. Here we compare the transduction pattern of neurons in the somatosensory system following injection of AAV9 or AAV2retro in the parabrachial complex of the midbrain, the spinal cord dorsal horn, the intrathecal space, and the colon. Transduction was evaluated based on Cre-dependent expression of tdTomato in transgenic reporter mice, following delivery of AAV9 or AAV2retro carrying identical constructs that drive the expression of Cre/GFP. The pattern of distribution of tdTomato expression indicated notable differences in the access of the two AAV serotypes to primary afferent neurons via peripheral delivery in the colon and to spinal projections neurons via intracranial delivery within the parabrachial complex. Additionally, our results highlight the superior sensitivity of detection of neuronal transduction based on reporter expression relative to expression of viral products.

Biodistribution of Adeno-Associated Virus Serotype 5 Viral Vectors Following Intrathecal Injection.

The pharmacokinetic profile of AAV particles following intrathecal delivery has not yet been clearly defined. The present study evaluated the distribution profile of adeno-associated virus serotype 5 (AAV5) viral vectors following lumbar intrathecal injection in mice. After a single bolus intrathecal injection, viral DNA concentrations in mouse whole blood, spinal cord, and peripheral tissues were determined using quantitative polymerase chain reaction (qPCR). The kinetics of AAV5 vector in whole blood and the concentration over time in spinal and peripheral tissues were analyzed. Distribution of the AAV5 vector to all levels of the spinal cord, dorsal root ganglia, and into systemic circulation occurred rapidly within 30 min following injection. Vector concentration in whole blood reached a maximum 6 h postinjection with a half-life of approximately 12 h. Area under the curve data revealed the highest concentration of vector distributed to dorsal root ganglia tissue. Immunohistochemical analysis revealed AAV5 particle colocalization with the pia mater at the spinal cord and macrophages in the dorsal root ganglia (DRG) 30 min after injection. These results demonstrate the widespread distribution of AAV5 particles through cerebrospinal fluid and preferential targeting of DRG tissue with possible clearance mechanisms via DRG macrophages.

Comparative Effectiveness of Intracerebroventricular, Intrathecal, and Intranasal Routes of AAV9 Vector Administration for Genetic Therapy of Neurologic Disease in Murine Mucopolysaccharidosis Type I.

Mucopolysaccharidosis type I (MPS I) is an inherited metabolic disorder caused by deficiency of the lysosomal enzyme alpha-L-iduronidase (IDUA). The two current treatments [hematopoietic stem cell transplantation (HSCT) and enzyme replacement therapy (ERT)], are insufficiently effective in addressing neurologic disease, in part due to the inability of lysosomal enzyme to cross the blood brain barrier. With a goal to more effectively treat neurologic disease, we have investigated the effectiveness of AAV-mediated IDUA gene delivery to the brain using several different routes of administration. Animals were treated by either direct intracerebroventricular (ICV) injection, by intrathecal (IT) infusion into the cerebrospinal fluid, or by intranasal (IN) instillation of AAV9-IDUA vector. AAV9-IDUA was administered to IDUA-deficient mice that were either immunosuppressed with cyclophosphamide (CP), or immunotolerized at birth by weekly injections of human iduronidase. In animals treated by ICV or IT administration, levels of IDUA enzyme ranged from 3- to 1000-fold that of wild type levels in all parts of the microdissected brain. In animals administered vector intranasally, enzyme levels were 100-fold that of wild type in the olfactory bulb, but enzyme expression was close to wild type levels in other parts of the brain. Glycosaminoglycan levels were reduced to normal in ICV and IT treated mice, and in IN treated mice they were normalized in the olfactory bulb, or reduced in other parts of the brain. Immunohistochemical analysis showed extensive IDUA expression in all parts of the brain of ICV treated mice, while IT treated animals showed transduction that was primarily restricted to the hind brain with some sporadic labeling seen in the mid- and fore brain. At 6 months of age, animals were tested for spatial navigation, memory, and neurocognitive function in the Barnes maze; all treated animals were indistinguishable from normal heterozygous control animals, while untreated IDUA deficient animals exhibited significant learning and spatial navigation deficits. We conclude that IT and IN routes are acceptable and alternate routes of administration, respectively, of AAV vector delivery to the brain with effective IDUA expression, while all three routes of administration prevent the emergence of neurocognitive deficiency in a mouse MPS I model.

Function of Renal Nerves in Kidney Physiology and Pathophysiology.

Renal sympathetic (efferent) nerves play an important role in the regulation of renal function, including glomerular filtration, sodium reabsorption, and renin release. The kidney is also innervated by sensory (afferent) nerves that relay information to the brain to modulate sympathetic outflow. Hypertension and other cardiometabolic diseases are linked to overactivity of renal sympathetic and sensory nerves, but our mechanistic understanding of these relationships is limited. Clinical trials of catheter-based renal nerve ablation to treat hypertension have yielded promising results. Therefore, a greater understanding of how renal nerves control the kidney under physiological and pathophysiological conditions is needed. In this review, we provide an overview of the current knowledge of the anatomy of efferent and afferent renal nerves and their functions in normal and pathophysiological conditions. We also suggest further avenues of research for development of novel therapies targeting the renal nerves.

The nAChR Chaperone TMEM35a (NACHO) Contributes to the Development of Hyperalgesia in Mice.

Pain is a major health problem, affecting over fifty million adults in the US alone, with significant economic cost in medical care and lost productivity. Despite evidence implicating nicotinic acetylcholine receptors (nAChRs) in pathological pain, their specific contribution to pain processing in the spinal cord remains unclear given their presence in both neuronal and non-neuronal cell types. Here we investigated if loss of neuronal-specific TMEM35a (NACHO), a novel chaperone for functional expression of the homomeric α7 and assembly of the heteromeric α3, α4, and α6-containing nAChRs, modulates pain in mice. Mice with tmem35a deletion exhibited thermal hyperalgesia and mechanical allodynia. Intrathecal administration of nicotine and the α7-specific agonist, PHA543613, produced analgesic responses to noxious heat and mechanical stimuli in tmem35a KO mice, respectively, suggesting residual expression of these receptors or off-target effects. Since NACHO is expressed only in neurons, these findings indicate that neuronal α7 nAChR in the spinal cord contributes to heat nociception. To further determine the molecular basis underlying the pain phenotype, we analyzed the spinal cord transcriptome. Compared to WT control, the spinal cord of tmem35a KO mice exhibited 72 differentially-expressed genes (DEGs). These DEGs were mapped onto functional gene networks using the knowledge-based database, Ingenuity Pathway Analysis, and suggests increased neuroinflammation as a potential contributing factor for the hyperalgesia in tmem35a KO mice. Collectively, these findings implicate a heightened inflammatory response in the absence of neuronal NACHO activity. Additional studies are needed to determine the precise mechanism by which NACHO in the spinal cord modulates pain.

Peripheral Deltorphin II Inhibits Nociceptors Following Nerve Injury.

Clinical and preclinical studies have revealed that local administration of opioid agonists into peripheral tissue attenuates inflammatory pain. However, few studies have examined whether peripherally restricted opioids are effective in reducing mechanical allodynia and hyperalgesia that usually follows nerve injury. The aim of the present study was to determine whether the mechanical responsiveness of C-fiber mechanical nociceptors innervating skin under neuropathic pain conditions is depressed by direct activation of delta opioid receptors (DORs) on their peripheral terminals. A murine model of peripheral neuropathic pain was induced with a spared nerve (tibial) injury, in which mice survived 7 or 28 days after surgery before electrophysiological testing began. Control groups comprised naïve and sham-operated animals. An ex vivo preparation of mouse plantar skin with attached tibial nerve was used to examine electrophysiologically the effects of the selective DOR agonist, deltorphin II, on the response properties of individual cutaneous C-fiber nociceptors. In contrast to naïve and sham-operated animals, deltorphin II induced an inhibition of the mechanical responsiveness of C-fiber mechanical nociceptors innervating skin under neuropathic conditions. The effects of deltorphin II were concentration-dependent and prevented by pretreatment with naltrindole indicating DOR-mediated inhibitory effects of deltorphin II. Our results provide the first direct evidence for expression of functional DORs on mechanical nociceptors innervating skin in an animal model of neuropathic pain.

Intravenous delivery for treatment of mucopolysaccharidosis type I: A comparison of AAV serotypes 9 and rh10.

Mucopolysaccharidosis type I (MPS I) is an inherited metabolic disorder caused by deficiency of alpha-L-iduronidase (IDUA), resulting in accumulation of heparan and dermatan sulfate glycosaminoglycans (GAGs). Individuals with the most severe form of the disease (Hurler syndrome) suffer from neurodegeneration, intellectual disability, and death by age 10. Current treatments for this disease include allogeneic hematopoietic stem cell transplantation (HSCT) and enzyme replacement therapy (ERT). However, these treatments do not address CNS manifestations of the disease. In this study we compared the ability of intravenously administered AAV serotypes 9 and rh10 (AAV9 and AAVrh10) for delivery and expression of the IDUA gene in the CNS. Adult C57BL/6 MPS I mice were infused intravenously with either AAV9 or AAVrh10 vector encoding the human IDUA gene. Treated animals demonstrated supraphysiological levels and widespread restoration of IDUA enzyme activity in the plasma and all organs including the CNS. High levels of IDUA enzyme activity were observed in the plasma, brain and spinal cord ranging from 10 to 100-fold higher than heterozygote controls, while levels in peripheral organs were also high, ranging from 1000 to 10,000-fold higher than control animals. In general, levels of IDUA expression were slightly higher in peripheral organs for AAVrh10 administered animals although these differences were not significant except for the lung. Levels of IDUA expression between AAV 9 and rh10 were roughly equivalent in the brain. Urinary and tissue GAGs were significantly reduced starting at 3 weeks after vector infusion, with restoration of normal GAG levels by the end of the study in animals treated with either AAV9 or rh10. These results demonstrate that non-invasive intravenous AAV9 or AAVrh10-mediated IDUA gene therapy is a potentially effective treatment for both systemic and CNS manifestations of MPS I, with implications for the treatment of other metabolic and neurological diseases as well.

Optogenetic-induced sympathetic neuromodulation of brown adipose tissue thermogenesis.

The brown adipose tissue (BAT) is a thermogenic organ that plays a major role in energy balance, obesity, and diabetes due to the potent glucose and lipid clearance that fuels its thermogenesis, which is largely mediated via sympathetic nervous system activation. However, thus far there has been little experimental validation of the hypothesis that selective neuromodulation of the sympathetic nerves innervating the BAT is sufficient to elicit thermogenesis in mice. We generated mice expressing blue light-activated channelrhodopsin-2 (ChR2) in the sympathetic nerves innervating the BAT using two different strategies: injecting the BAT of C57Bl/6J mice with AAV6-hSyn-ChR2 (H134R)-EYFP; crossbreeding tyrosine hydroxylase-Cre mice with floxed-stop ChR2-EYFP mice. The nerves in the BAT expressing ChR2 were selectively stimulated with a blue LED light positioned underneath the fat pad of anesthetized mice, while the BAT and core temperatures were simultaneously recorded. Using immunohistochemistry we confirmed the selective expression of EYFP in TH positive nerves fibers. In addition, local optogenetic stimulation of the sympathetic nerves induced significant increase in the BAT temperature followed by an increase in core temperature in mice expressing ChR2, but not in the respective controls. The BAT activation was also paralleled by increased levels of pre-UCP1 transcript. Our results demonstrate that local optogenetic stimulation of the sympathetic nerves is sufficient to elicit BAT and core thermogenesis, thus suggesting that peripheral neuromodulation has the potential to be exploited as an alternative to pharmacotherapies to elicit organ activation and thus ameliorate type 2 diabetes and/or obesity.

AAV-Mediated Gene Delivery to the Spinal Cord by Intrathecal Injection.

Gene therapy targeting the spinal cord is an important tool for analyzing mechanisms of nervous system diseases and the development of gene therapies. Analogous to a lumbar puncture in humans, the rodent spinal cord can be accessed through an efficient, noninvasive injection. Here we describe a method for AAV-mediated gene transfer to cells of the spinal cord by intrathecal injection of small quantities of AAV vector.

AAV-Mediated Gene Delivery to the Enteric Nervous System by Intracolonic Injection.

The enteric nervous system of the lower gastrointestinal tract comprises intrinsic neural circuits as well as extrinsic afferent and efferent innervation. The development of strategies for neuronal gene transfer has created new opportunities for functional analysis, circuit mapping, and neuromodulation in the enteric nervous system. Studies of AAV-mediated gene transfer to enteric neurons and dorsal root ganglion neurons (DRG) have provided proofs-of-concept for the utility of AAV vectors for genetic manipulations of the intrinsic and extrinsic components of the enteric nervous system. Here we describe a method for AAV-mediated gene transfer to enteric neurons of the descending colon as well as colon-innervating DRG neurons by injection within the intestinal wall (intracolonic injection).

Detailed Method for Intrathecal Delivery of Gene Therapeutics by Direct Lumbar Puncture in Mice.

Delivery of viral vectors directly into the central nervous system (CNS) has emerged as an important tool for the refinement of gene therapy. Intrathecal delivery by direct lumbar puncture in conscious rodents offers a minimally invasive approach that avoids tissue damage and/or destruction. Here we describe delivery of small quantities of viral vector product to the intrathecal space of rodents via direct lumbar puncture aided by a catheter.

The purinergic receptor P2RX7 directs metabolic fitness of long-lived memory CD8+ T cells.

Extracellular ATP (eATP) is an ancient 'danger signal' used by eukaryotes to detect cellular damage1. In mice and humans, the release of eATP during inflammation or injury stimulates both innate immune activation and chronic pain through the purinergic receptor P2RX72-4. It is unclear, however, whether this pathway influences the generation of immunological memory, a hallmark of the adaptive immune system that constitutes the basis of vaccines and protective immunity against re-infection5,6. Here we show that P2RX7 is required for the establishment, maintenance and functionality of long-lived central and tissue-resident memory CD8+ T cell populations in mice. By contrast, P2RX7 is not required for the generation of short-lived effector CD8+ T cells. Mechanistically, P2RX7 promotes mitochondrial homeostasis and metabolic function in differentiating memory CD8+ T cells, at least in part by inducing AMP-activated protein kinase. Pharmacological inhibitors of P2RX7 provoked dysregulated metabolism and differentiation of activated mouse and human CD8+ T cells in vitro, and transient P2RX7 blockade in vivo ameliorated neuropathic pain but also compromised production of CD8+ memory T cells. These findings show that activation of P2RX7 by eATP provides a common currency that both alerts the nervous and immune system to tissue damage, and promotes the metabolic fitness and survival of the most durable and functionally relevant memory CD8+ T cell populations.

Involvement of the VGF-derived peptide TLQP-62 in nerve injury-induced hypersensitivity and spinal neuroplasticity.

Neuroplasticity in the dorsal horn after peripheral nerve damage contributes critically to the establishment of chronic pain. The neurosecretory protein VGF (nonacronymic) is rapidly and robustly upregulated after nerve injury, and therefore, peptides generated from it are positioned to serve as signals for peripheral damage. The goal of this project was to understand the spinal modulatory effects of the C-terminal VGF-derived peptide TLQP-62 at the cellular level and gain insight into the function of the peptide in the development of neuropathic pain. In a rodent model of neuropathic pain, we demonstrate that endogenous levels of TLQP-62 increased in the spinal cord, and its immunoneutralization led to prolonged attenuation of the development of nerve injury-induced hypersensitivity. Using multiphoton imaging of submaximal glutamate-induced Ca responses in spinal cord slices, we demonstrate the ability of TLQP-62 to potentiate glutamatergic responses in the dorsal horn. We further demonstrate that the peptide selectively potentiates responses of high-threshold spinal neurons to mechanical stimuli in singe-unit in vivo recordings. These findings are consistent with a function of TLQP-62 in spinal plasticity that may contribute to central sensitization after nerve damage.

Bivalent ligand that activates mu opioid receptor and antagonizes mGluR5 receptor reduces neuropathic pain in mice.

The mu opioid receptor (MOR) and metabotropic glutamate receptor 5 (mGluR5) are well-established pharmacological targets in the management of chronic pain. Both receptors are expressed in the spinal cord. MMG22, a bivalent ligand containing 2 pharmacophores separated by 22 atoms, which simultaneously activates MOR and antagonizes mGluR5, has been shown to produce potent reversal of tactile hypersensitivity in rodent models of lipopolysaccharide (LPS)-and bone cancer-induced chronic pain. This study assessed whether intrathecal MMG22 also is effective in reducing pain of neuropathic origin. Furthermore, we theorized that MMG22 should reduce hyperalgesia in nerve-injured mice in a manner consistent with a synergistic interaction between MOR and mGluR5. Several weeks after spared nerve injury, tactile hypersensitivity was reversed in mice by the intrathecal injection of MMG22 (0.01-10 nmol) but also by its shorter spacer analog, MMG10, with similar potency. The potencies of the bivalent ligands were 10- to 14-fold higher than those of the compounds upon which the bivalent structure was based, the MOR agonist oxymorphone and the mGluR5 antagonist MPEP. Coadministration of oxymorphone and MPEP demonstrated analgesic synergism, an interaction confirmed by isobolographic analysis. This study indicates that in the spared nerve injury-induced model of neuropathic pain, the 2 pharmacophores of the bivalent ligands MMG22 and MMG10 target MOR and mGluR5 as separate receptor monomers. The observed increase in the potency of MMG22 and MMG10, compared with oxymorphone and MPEP, may reflect the synergistic interaction of the 2 pharmacophores of the bivalent ligand acting at their respective separate receptor monomers.