In-Cell Penetration Selection-Mass Spectrometry Produces Noncanonical Peptides for Antisense Delivery.

Peptide-mediated delivery of macromolecules in cells has significant potential therapeutic benefits, but no therapy employing cell-penetrating peptides (CPPs) has reached the market after 30 years of investigation due to challenges in the discovery of new, more efficient sequences. Here, we demonstrate a method for in-cell penetration selection-mass spectrometry (in-cell PS-MS) to discover peptides from a synthetic library capable of delivering macromolecule cargo to the cytosol. This method was inspired by recent in vivo selection approaches for cell-surface screening, with an added spatial dimension resulting from subcellular fractionation. A representative peptide discovered in the cytosolic extract, Cyto1a, is nearly 100-fold more active toward antisense phosphorodiamidate morpholino oligomer (PMO) delivery compared to a sequence identified from a whole cell extract, which includes endosomes. Cyto1a is composed of d-residues and two non-α-amino acids, is more stable than its all-l isoform, and is less toxic than known CPPs with comparable activity. Pulse-chase and microscopy experiments revealed that while the PMO-Cyto1a conjugate is likely taken up by endosomes, it can escape to localize to the nucleus without nonspecifically releasing other endosomal components. In-cell PS-MS introduces a means to empirically discover unnatural synthetic peptides for subcellular delivery of therapeutically relevant cargo.

Cell-Penetrating d-Peptides Retain Antisense Morpholino Oligomer Delivery Activity.

Cell-penetrating peptides (CPPs) can cross the cell membrane to enter the cytosol and deliver otherwise nonpenetrant macromolecules such as proteins and oligonucleotides. For example, recent clinical trials have shown that a CPP attached to phosphorodiamidate morpholino oligomers (PMOs) resulted in higher muscle concentration, increased exon skipping, and dystrophin production relative to another study of the PMO alone in patients of Duchenne muscular dystrophy. Therefore, effective design and the study of CPPs could help enhance therapies for difficult-to-treat diseases. So far, the study of CPPs for PMO delivery has been restricted to predominantly canonical l-peptides. We hypothesized that mirror-image d-peptides could have similar PMO delivery activity as well as enhanced proteolytic stability, facilitating their characterization and quantification from biological milieu. We found that several enantiomeric peptide sequences could deliver a PMO-biotin cargo with similar activities while remaining stable against serum proteolysis. The biotin label allowed for affinity capture of fully intact PMO-peptide conjugates from whole-cell and cytosolic lysates. By profiling a mixture of these constructs in cells, we determined their relative intracellular concentrations. When combined with PMO activity, these concentrations provide a new metric for delivery efficiency, which may be useful for determining which peptide sequence to pursue in further preclinical studies.

Deep Learning Enables Discovery of a Short Nuclear Targeting Peptide for Efficient Delivery of Antisense Oligomers.

Therapeutic macromolecules such as proteins and oligonucleotides can be highly efficacious but are often limited to extracellular targets due to the cell's impermeable membrane. Cell-penetrating peptides (CPPs) are able to deliver such macromolecules into cells, but limited structure-activity relationships and inconsistent literature reports make it difficult to design effective CPPs for a given cargo. For example, polyarginine motifs are common in CPPs, promoting cell uptake at the expense of systemic toxicity. Machine learning may be able to address this challenge by bridging gaps between experimental data in order to discern sequence-activity relationships that evade our intuition. Our earlier data set and deep learning model led to the design of miniproteins (>40 amino acids) for antisense delivery. Here, we leveraged and expanded our model with data augmentation in the short CPP sequence space of the data set to extrapolate and discover short, low-arginine-content CPPs that would be easier to synthesize and amenable to rapid conjugation to desired cargo, and with minimal in vivo toxicity. The lead predicted peptide, termed P6, is as active as a polyarginine CPP for the delivery of an antisense oligomer, while having only one arginine side chain and 18 total residues. We determined the pentalysine motif and the C-terminal cysteine of P6 to be the main drivers of activity. The antisense conjugate was able to enhance corrective splicing in an animal model to produce functional eGFP in heart tissue in vivo while remaining nontoxic up to a dose of 60 mg/kg. In addition, P6 was able to deliver an enzyme to the cytosol of cells. Our findings suggest that, given a data set of long CPPs, we can discover by extrapolation short, active sequences that deliver antisense oligomers.

Deep learning to design nuclear-targeting abiotic miniproteins.

There are more amino acid permutations within a 40-residue sequence than atoms on Earth. This vast chemical search space hinders the use of human learning to design functional polymers. Here we show how machine learning enables the de novo design of abiotic nuclear-targeting miniproteins to traffic antisense oligomers to the nucleus of cells. We combined high-throughput experimentation with a directed evolution-inspired deep-learning approach in which the molecular structures of natural and unnatural residues are represented as topological fingerprints. The model is able to predict activities beyond the training dataset, and simultaneously deciphers and visualizes sequence-activity predictions. The predicted miniproteins, termed 'Mach', reach an average mass of 10 kDa, are more effective than any previously known variant in cells and can also deliver proteins into the cytosol. The Mach miniproteins are non-toxic and efficiently deliver antisense cargo in mice. These results demonstrate that deep learning can decipher design principles to generate highly active biomolecules that are unlikely to be discovered by empirical approaches.

Inhibition of Inactive States of Tetrodotoxin-Sensitive Sodium Channels Reduces Spontaneous Firing of C-Fiber Nociceptors and Produces Analgesia in Formalin and Complete Freund's Adjuvant Models of Pain.

While genetic evidence shows that the Nav1.7 voltage-gated sodium ion channel is a key regulator of pain, it is unclear exactly how Nav1.7 governs neuronal firing and what biophysical, physiological, and distribution properties of a pharmacological Nav1.7 inhibitor are required to produce analgesia. Here we characterize a series of aminotriazine inhibitors of Nav1.7 in vitro and in rodent models of pain and test the effects of the previously reported "compound 52" aminotriazine inhibitor on the spiking properties of nociceptors in vivo. Multiple aminotriazines, including some with low terminal brain to plasma concentration ratios, showed analgesic efficacy in the formalin model of pain. Effective concentrations were consistent with the in vitro potency as measured on partially-inactivated Nav1.7 but were far below concentrations required to inhibit non-inactivated Nav1.7. Compound 52 also reversed thermal hyperalgesia in the complete Freund's adjuvant (CFA) model of pain. To study neuronal mechanisms, electrophysiological recordings were made in vivo from single nociceptive fibers from the rat tibial nerve one day after CFA injection. Compound 52 reduced the spontaneous firing of C-fiber nociceptors from approximately 0.7 Hz to 0.2 Hz and decreased the number of action potentials evoked by suprathreshold tactile and heat stimuli. It did not, however, appreciably alter the C-fiber thresholds for response to tactile or thermal stimuli. Surprisingly, compound 52 did not affect spontaneous activity or evoked responses of Aδ-fiber nociceptors. Results suggest that inhibition of inactivated states of TTX-S channels, mostly likely Nav1.7, in the peripheral nervous system produces analgesia by regulating the spontaneous discharge of C-fiber nociceptors.

Global Nav1.7 knockout mice recapitulate the phenotype of human congenital indifference to pain.

Clinical genetic studies have shown that loss of Nav1.7 function leads to the complete loss of acute pain perception. The global deletion is reported lethal in mice, however, and studies of mice with promoter-specific deletions of Nav1.7 have suggested that the role of Nav1.7 in pain transduction depends on the precise form of pain. We developed genetic and animal husbandry strategies that overcame the neonatal-lethal phenotype and enabled construction of a global Nav1.7 knockout mouse. Knockouts were anatomically normal, reached adulthood, and had phenotype wholly analogous to human congenital indifference to pain (CIP): compared to littermates, knockouts showed no defects in mechanical sensitivity or overall movement yet were completely insensitive to painful tactile, thermal, and chemical stimuli and were anosmic. Knockouts also showed no painful behaviors resulting from peripheral injection of nonselective sodium channel activators, did not develop complete Freund's adjuvant-induced thermal hyperalgesia, and were insensitive to intra-dermal histamine injection. Tetrodotoxin-sensitive sodium current recorded from cell bodies of isolated sensory neurons and the mechanically-evoked spiking of C-fibers in a skin-nerve preparation each were reduced but not eliminated in tissue from knockouts compared to littermates. Results support a role for Nav1.7 that is conserved between rodents and humans and suggest several possibly translatable biomarkers for the study of Nav1.7-targeted therapeutics. Results further suggest that Nav1.7 may retain its key role in persistent as well as acute forms of pain.

Discovery and optimization of aminopyrimidinones as potent and state-dependent Nav1.7 antagonists.

Clinical genetic data have shown that the product of the SCN9A gene, voltage-gated sodium ion channel Nav1.7, is a key control point for pain perception and a possible target for a next generation of analgesics. Sodium channels, however, historically have been difficult drug targets, and many of the existing structure-activity relationships (SAR) have been defined on pharmacologically modified channels with indirect reporter assays. Herein we describe the discovery, optimization, and SAR of potent aminopyrimidinone Nav1.7 antagonists using electrophysiology-based assays that measure the ligand-receptor interaction directly. Within this series, rapid functionalization at the polysubstituted aminopyrimidinone head group enabled exploration of SAR and of pharmacokinetic properties. Lead optimized N-Me-aminopyrimidinone 9 exhibited improved Nav1.7 potency, minimal off-target hERG liability, and improved rat PK properties.

Identification of a potent, state-dependent inhibitor of Nav1.7 with oral efficacy in the formalin model of persistent pain.

Clinical human genetic studies have recently identified the tetrodotoxin (TTX) sensitive neuronal voltage gated sodium channel Nav1.7 (SCN9A) as a critical mediator of pain sensitization. Herein, we report structure-activity relationships for a novel series of 2,4-diaminotriazines that inhibit hNav1.7. Optimization efforts culminated in compound 52, which demonstrated pharmacokinetic properties appropriate for in vivo testing in rats. The binding site of compound 52 on Nav1.7 was determined to be distinct from that of local anesthetics. Compound 52 inhibited tetrodotoxin-sensitive sodium channels recorded from rat sensory neurons and exhibited modest selectivity against the hERG potassium channel and against cloned and native tetrodotoxin-resistant sodium channels. Upon oral administration to rats, compound 52 produced dose- and exposure-dependent efficacy in the formalin model of pain.

Pharmacological effects of nonselective and subtype-selective nicotinic acetylcholine receptor agonists in animal models of persistent pain.

Nicotinic acetylcholine receptors (nAChRs) are longstanding targets for a next generation of pain therapeutics, but the nAChR subtypes that govern analgesia remain unknown. We tested a series of nicotinic agonists, including many molecules used or tried clinically, on a panel of cloned neuronal nAChRs for potency and selectivity using patch-clamp electrophysiology and a live cell-based fluorescence assay. Nonselective nicotinic agonists as well as compounds selective either for alpha4beta2 or for alpha7 nAChRs were then tested in the formalin and complete Freund's adjuvant models of pain. Nonselective nAChR agonists ABT-594 and varenicline were effective analgesics. By contrast, the selective alpha4beta2 agonist ispronicline and a novel alpha4beta2-selective potentiator did not appear to produce analgesia in either model. alpha7-selective agonists reduced the pain-related endpoint, but the effect could be ascribed to nonspecific reduction of movement rather than to analgesia. Neither selective nor nonselective alpha7 nicotinic agonists affected the release of pro-inflammatory cytokines in response to antigen challenge. Electrophysiological recordings from spinal cord slice showed a strong nicotine-induced increase in inhibitory synaptic transmission that was mediated partially by alpha4beta2 and only minimally by alpha7 subtypes. Taken with previous studies, the results suggest that agonism of alpha4beta2 nAChRs is necessary but not sufficient to produce analgesia, and that the spinal cord is a key site where the molecular action of nAChRs produces analgesia.

Discovery and optimization of substituted piperidines as potent, selective, CNS-penetrant alpha4beta2 nicotinic acetylcholine receptor potentiators.

The discovery of a series of small molecule alpha4beta2 nAChR potentiators is reported. The structure-activity relationship leads to potent compounds selective against nAChRs including alpha3beta2 and alpha3beta4 and optimized for CNS penetrance. Compounds increased currents through recombinant alpha4beta2 nAChRs, yet did not compete for binding with the orthosteric ligand cytisine. High potency and efficacy on the rat channel combined with good PK properties will allow testing of the alpha4beta2 potentiator mechanism in animal models of disease.

Synthesis and activity of substituted carbamates as potentiators of the alpha4beta2 nicotinic acetylcholine receptor.

The synthesis and structure-activity relationship of a series of carbamate potentiators of alpha4beta2 nAChR is reported herein. These compounds were highly selective for alpha4beta2 over other nAChR subtypes. In addition, compounds increased the response of alpha4beta2 nAChRs to acetylcholine, as measured with patch-clamp electrophysiology.

Pharmacological, pharmacokinetic, and primate analgesic efficacy profile of the novel bradykinin B1 Receptor antagonist ELN441958.

The bradykinin B(1) receptor plays a critical role in chronic pain and inflammation, although efforts to demonstrate efficacy of receptor antagonists have been hampered by species-dependent potency differences, metabolic instability, and low oral exposure of current agents. The pharmacology, pharmacokinetics, and analgesic efficacy of the novel benzamide B(1) receptor antagonist 7-chloro-2-[3-(9-pyridin-4-yl-3,9-diazaspiro[5.5]undecanecarbonyl)phenyl]-2,3-dihydro-isoindol-1-one (ELN441958) is described. ELN441958 competitively inhibited the binding of the B(1) agonist ligand [(3)H]desArg(10)-kallidin ([(3)H]DAKD) to IMR-90 human fibroblast membranes with high affinity (K(i) = 0.26 +/- 0.02 nM). ELN441958 potently antagonized DAKD (but not bradykinin)-induced calcium mobilization in IMR-90 cells, indicating that it is highly selective for B(1) over B(2) receptors. Antagonism of agonist-induced calcium responses at B(1) receptors from different species indicated that ELN441958 is selective for primate over rodent B(1) receptors with a rank order potency (K(B), nanomolar) of human (0.12 +/- 0.02) approximately rhesus monkey (0.24 +/- 0.01) > rat (1.5 +/- 0.4) > mouse (14 +/- 4). ELN441958 had good permeability and metabolic stability in vitro consistent with high oral exposure and moderate plasma half-lives in rats and rhesus monkeys. Because ELN441958 is up to 120-fold more potent at primate than at rodent B(1) receptors, it was evaluated in a primate pain model. ELN441958 dose-dependently reduced carrageenan-induced thermal hyperalgesia in a rhesus monkey tail-withdrawal model, with an ED(50) approximately 3 mg/kg s.c. Naltrexone had no effect on the antihyperalgesia produced by ELN441958, indicating a lack of involvement of opioid receptors. ELN441958 is a novel small molecule bradykinin B(1) receptor antagonist exhibiting high oral bioavailability and potent systemic efficacy in rhesus monkey inflammatory pain.

Models of nociception: hot-plate, tail-flick, and formalin tests in rodents.

Experimental models of pain include tests of response thesholds to high intensity stimuli (acute pain tests) and changes in spontaneous or evoked behavioral responses in animals with peripheral injury or inflammation (persistent pain models). Acute thermal pain is modeled by the hot-plate and tail-flick test, while persistent pain can be modeled by the formalin test. This unit presents protocols for all three of these tests, including preparation of animals (rats or mice), administration of a compound being tested for its analgesic properties and data collection.

Impaired formalin-evoked changes of spinal amino acid levels in diabetic rats.

To investigate mechanisms by which diabetes alters sensory processing, we measured levels of amino acid neurotransmitters in spinal dialysates from awake, unrestrained control and diabetic rats under resting conditions and following hind paw formalin injection. Under resting conditions, glutamate concentrations in spinal dialysates were significantly (P<0.05) decreased in diabetic rats compared to those of control rats whereas aspartate, taurine, glycine and citrulline remained unchanged and GABA was significantly (P<0.05) increased. Noxious stimulation of the hind paw by subcutaneous injection of 0.5% formalin into the dorsum caused a defined flinching behavior in the afflicted paw, and the amount of flinching was significantly (P<0.05) greater in diabetic rats than in controls. Paw formalin injection significantly (P<0.05) increased dialysate levels of glutamate, aspartate, taurine, glycine and citrulline by 3- to 4-fold above basal in both control and diabetic rats. The concentration of glutamate in dialysate samples collected immediately after paw formalin injection remained significantly (P<0.05) lower in diabetic rats compared to those in controls. Formalin injection did not alter dialysate GABA concentrations in control rats, whereas in diabetic rats there was an increase of 151+/-15% above basal levels. These findings indicate that the selective depression of basal and stimulus-evoked glutamate levels in the spinal cord of diabetic rats occurs in parallel with elevated spinal GABA levels. Because increased pain-associated behavior is accompanied by an attenuated spinal glutamate spike following paw formalin injection, hyperalgesia in diabetic rats does not appear to be secondary to enhanced glutamatergic input to the spinal cord.

Molecular pain, a new era of pain research and medicine.

Molecular pain is a relatively new and rapidly expanding research field that represents an advanced step from conventional pain research. Molecular pain research addresses physiological and pathological pain at the cellular, subcellular and molecular levels. These studies integrate pain research with molecular biology, genomics, proteomics, modern electrophysiology and neurobiology. The field of molecular pain research has been rapidly expanding in the recent years, and has great promise for the identification of highly specific and effective targets for the treatment of intractable pain. Although several existing journals publish articles on classical pain research, none are specifically dedicated to molecular pain research. Therefore, a new journal focused on molecular pain research is needed. Molecular Pain, an Open Access, peer-reviewed, online journal, will provide a forum for molecular pain scientists to communicate their research findings in a targeted manner to others in this important and growing field.

Reduced heat sensitivity and epidermal nerve fiber immunostaining following single applications of a high-concentration capsaicin patch.

Capsaicin-containing plant extracts have been used as topical treatments for a variety of pain syndromes for many centuries. Current products containing capsaicin in low concentrations (usually 0.025-0.075% w/w) have shown efficacy against a variety of pain conditions in clinical studies. However, in order to produce significant analgesic effects, these formulations require frequent re-dosing, often as much as three to five times daily for several weeks. Previous functional and immunohistochemical studies following prolonged exposures to low-concentration capsaicin cream suggested that the duration and onset of analgesic efficacy correlate with a reduction of cutaneous nociceptive sensory nerve fiber responsiveness and immunostaining. The purpose of the present study was to determine whether a single topical application of a high-concentration capsaicin-containing (8%w/w) patch for 120 min or less would induce similar effects on cutaneous nociceptive nerve fibers. Seven days following patch application, changes in heat and cold perception thresholds were determined by quantitative sensory testing and punch biopsies were collected to assess epidermal nerve fiber (ENF) immunostaining density at the application site using PGP 9.5 as a marker. The results show a significant reduction of heat, but not cold, sensitivity and reduction of ENF immunostaining with high-capsaicin concentration patch applications for 60 or 120 min, compared to placebo patch applications. Application sites exposed to low-capsaicin concentration (0.04%w/w) patches for 120 min or high-concentration patches for 30 min were not significantly different from placebo with respect to either thermal threshold detection or ENF immunostaining. The ability of a single 60 min high-concentration patch application to mimic effects produced by prolonged exposure to low-concentration capsaicin creams suggests a new approach to the management of chronic pain syndromes.

Powerful antinociceptive effects of the cone snail venom-derived subtype-selective NMDA receptor antagonists conantokins G and T.

Subunit non-selective N-methyl-D-aspartate (NMDA) receptor antagonists reduce injury-induced pain behavior, but generally produce unacceptable side effects. In this study, we examined the antinociceptive and motor effects of cone snail venom-derived peptides, conantokins G and T (conG and conT), which are selective inhibitors of the NR2B or NR2A and NR2B subtypes of the NMDA receptor, respectively. We tested the effects of conG and conT in models of tissue (formalin test), nerve injury (partial sciatic nerve ligation) and inflammation-induced (intraplantar Complete Freund's Adjuvant; CFA) pain in mice. In the formalin test, intrathecal (i.t.) conG or conT suppressed the ongoing pain behavior (ED(50) and 95% confidence intervals (CI), 11 (7-19) and 19 (11-33), respectively) at doses that were 17-27 times lower than those required to impair motor function (accelerating rotarod treadmill test: ED(50) and 95% CI, 300 (120-730) and 320 (190-540) pmol, respectively). By comparison, SNX-111, an N-type voltage-sensitive calcium channel antagonist that is also derived from cone snail venom, produced significant motor impairment at a dose (3.0 pmol, i.t.) that was only partially efficacious in the formalin test. Furthermore, conG reversed the allodynia produced by nerve injury, with greater potency on thermal (ED50 and 95% CI, 24 (10-55) pmol) than on mechanical allodynia (59 (33-105) pmol). Finally, a single dose of conG (100 pmol, i.t.) also reduced CFA-evoked thermal and mechanical allodynia. Taken together, these results demonstrate that conantokins exhibit potent antinociceptive effects in several models of injury-induced pain. The study supports the notion that drugs directed against subtypes of the NMDA receptor, by virtue of their reduced side-effect profile, hold promise as novel therapeutic agents for the control of pain.

Elevated spinal cyclooxygenase and prostaglandin release during hyperalgesia in diabetic rats.

Diabetic rats display exaggerated hyperalgesic behavior in response to noxious stimuli that may model aspects of painful diabetic neuropathy. This study examined the contribution of spinal prostaglandin production to this exaggerated hyperalgesic behavior. Rats were implanted with spinal dialysis probes and received noxious stimulation to the hind paw by subcutaneous injection of 0.5% formalin solution. Prostaglandin E(2) (PGE(2)) was measured in dialysates of lumbar spinal cerebrospinal fluid concurrent with behavioral responses to formalin injection. In separate experiments, formalin-evoked behavioral responses were measured after intrathecal delivery of either a cyclooxygenase inhibitor or an EP(1) receptor antagonist, and cyclooxygenase protein was measured in spinal cord homogenates. Diabetic rats exhibited exaggerated behavioral responses to paw formalin injection and a concurrent prolongation of formalin-evoked PGE(2) release. Formalin-evoked behavioral responses were dose-dependently reduced in diabetic rats by spinal delivery of a cyclooxygenase inhibitor or an EP(1) receptor antagonist. Protein levels of cyclooxygenase-2 were elevated in the spinal cord of diabetic rats, whereas cyclooxygenase-1 protein was reduced. Hyperalgesic behavior in diabetic rats is associated with both increased cyclooxygenase-2 protein and cyclooxygenase-mediated PGE(2) release. Spinal delivery of selective inhibitors of cyclooxygenase-2 or antagonists of prostaglandin receptors may have therapeutic potential for treating painful diabetic neuropathy.

The 5-HT3 subtype of serotonin receptor contributes to nociceptive processing via a novel subset of myelinated and unmyelinated nociceptors.

Serotonin is a major component of the inflammatory chemical milieu and contributes to the pain of tissue injury via an action on multiple receptor subtypes. Here we studied mice after genetic or pharmacological disruption of the 5-HT(3) receptor, an excitatory serotonin-gated ion channel. We demonstrate that tissue injury-induced persistent, but not acute, nociception is significantly reduced after functional elimination of this receptor subtype. Specifically, in the setting of tissue injury, the 5-HT(3) receptor mediates activation of nociceptors but does not contribute to injury-associated edema. This result is explained by the localization of 5-HT(3) receptor transcripts to a previously uncharacterized subset of myelinated and unmyelinated afferents, few of which express the proinflammatory neuropeptide substance P. Finally, we provide evidence that central serotonergic circuits modulate nociceptive transmission via a facilitatory action at spinal 5-HT(3) receptors. We conclude that activation of both peripheral and central 5-HT(3) receptors is pronociceptive and that the contribution of peripheral 5-HT(3) receptors involves a novel complement of primary afferent nociceptors.

Reduced development of tolerance to the analgesic effects of morphine and clonidine in PKC gamma mutant mice.

A variety of second messenger systems have been implicated in the intracellular mechanisms of tolerance development to the analgesic actions of morphine, a mu opioid, and clonidine, an alpha-2 adrenergic receptor agonist. Here, we studied mice that carry a null mutation in the gene encoding a neuronal specific isoform of protein kinase C (PKC), namely, PKC gamma. We used the tail-flick test to construct dose-response curves before and 4 days after chronic morphine (75-mg pellets, subcutaneously (s.c.)) or clonidine treatment (0.3mg/kg, s.c., twice daily). Baseline tail-flick latencies did not differ in PKC gamma mutant and wild-type mice (3-4s). Both morphine and clonidine produced a dose-dependent suppression of the tail-flick response with an ED(50) (effective dose resulting in a 50% reduction of the control response) value (2.0mg/kg for morphine and 0.1mg/kg for clonidine) that was similar for naive mutant and wild-type mice. In contrast, after 4 days of drug delivery, mutant mice showed significantly less rightward shift in the dose-response curve to morphine (six-fold for wild-type and three-fold for mutant mice) and to clonidine (five-fold for wild-type and no shift for the mutant mice). These results indicate that PKC gamma contributes to the development of tolerance to the analgesic effects of both morphine and clonidine. Chronic morphine treatment can also result in sensitization of spinal cord neurons and increased pain behaviors following a noxious insult. To assess the contribution of PKC gamma to this process, we studied the responses of wild-type and mutant mice to an intraplantar injection of formalin (a model of persistent pain) following chronic morphine treatment. Although morphine tolerance increased formalin-evoked persistent pain behavior and Fos-LI in wild-type mice, there was no difference between placebo- and morphine-treated mutant mice, suggesting that PKC gamma also contributes to chronic morphine-induced changes in nociceptive processing.