Pain-related Behaviors In Mice Research Articles

Tacrolimus Induces Pain by Upregulating the Synaptic Expression of AMPA Receptors in the Spinal Cord Dorsal Horn

To investigate the effects of long-term administration of tacrolimus (also known as FK506) on the pain-related behaviors in mice and to study the underlying mechanism of pain induced by FK506 via measuring the effect of FK506 on the synaptic expression and phosphorylation of alpha-amino-3-hyroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor in the spinal cord dorsal horn of mice. 1) A total of 24 mice were evenly and randomly assigned to two groups, a FK506 group and a Saline group. The FK506 group was given daily intraperitoneal injection of FK506 and the Saline group received normal saline. Both groups received injection once a day for 7 days in a row. Some of the mice ( n=6 in each group) were monitored for the changes in the paw withdrawal threshold (PWT), the paw withdrawal latency (PWL), and the spontaneous pain behaviors to establish the pain model. The other mice ( n=6 in each group) of each group underwent isolation of the dorsal horn when obvious pain symptoms were induced on day 7 of injection. Then, immunoblotting was performed to determine the synaptic expression and phosphorylation levels of GluA1 and GluA2 subunits of AMPA receptors. 2) The mice were randomly divided into two groups, FK506+calcineurin (CaN) group and FK506+Saline group ( n=6 in each group). After the pain model was constructed, the mice were given intrathecal injection of recombinant CaN (also know as 33 U) or normal saline. Then, 60 minutes later, the PWT and the PWL of the mice were measured to investigate the role of CaN in FK506-induced pain. 3) Another18 mice were selected. The mice were randomly and evenly assigned to three groups, a control group (receiving intraperitoneal injection of normal saline followed by intrathecal injection of normal saline), FK506+Saline group (receiving intraperitoneal injection of FK506 followed by intrathecal injection of normal saline) and FK506+CaN group (receiving intraperitoneal injection of FK506 followed by intrathecal injection of CaN). Then, 60 minutes later, the spinal cords were isolated and subjected to immunoblotting assay to determine the role of CaN in FK506-induced AMPA receptor modification. 1) After 7 consecutive days of intraperitoneal injection of FK506, the PWT and PWL of mice dropped significantly, reaching on day 7 as low as 22.3%±0.05% and 66.6%±0.05% of the control group, respectively ( P<0.01). The FK506-treated mice displayed evident spontaneous pain behavior, presenting significantly increased licking activities ( P<0.01). These results indicated that FK506-induced pain model was successfully established. Immunoblotting assay showed that the total expressions of GluA1 and GluA2 subunits in the spinal dorsal horn of the FK506 group remained unchanged in comparison with those of the Saline group. However, FK506 specifically induced an increase in the synaptic expression of GluA1. In addition, the phosphorylation levels of GluA1 at Ser845 and Ser831 in FK506-treated mice were significantly increased in comparison with those of the control group ( P<0.05). 2) Compared with those of the mice in the FK506+Saline group, the PWT and the PWL of mice in the FK506+CaN group were significantly increased ( P<0.05). 3) Compared with those of the FK506+Saline group, the synaptic expression of GluA1 were decreased in FK506+CaN group ( P<0.01) and the phosphorylation levels of GluA1 at Ser845 and Ser831 were significantly downregulated ( P<0.001). The hyper-expression and hyperphosphorylation of GluA1 subunit in the spinal cord dorsal horn resulting from CaN inhibition contributes to the FK506-induced pain syndrome. FK506 induces the synaptic hyper-expression and hyperphosphorylation of GluA1 in the dorsal horn of the spinal cord through CaN inhibition, thereby inducing pain.

Region-specific involvement of ventral striatal dopamine D2 receptor-expressing medium spiny neurons in nociception

The ventrolateral striatum (VLS), a subregion of the ventral striatum (VS), possesses distinct neuronal Ca2+ activities and functions in reward-oriented behavior, compared with the ventromedial striatum (VMS) based on the anatomical feature. We hypothesized that the VLS exhibits unique neuronal activity and function in nociceptive processing, a part of aversive processing. Using fiber photometry to monitor the neuronal Ca2+ activities, we demonstrated that acute noxious mechanical stimuli like tail-pinch increased the Ca2+ activity of dopamine D2 receptor-expressing medium spiny neurons (D2-MSNs) in the VLS in correlation with the stimulus intensities in mice, whereas mechanical stimuli increased the VMS D2-MSN activity independent of the stimulus intensities. Likewise, thermal stimuli decreased the VLS and VMS D2-MSN Ca2+ activities during nociceptive behaviors in the hot plate test. Furthermore, the VLS D2-MSNs increased their Ca2+ activity accompanied by formalin-induced nociceptive behaviors in mice, whereas the VMS D2-MSNs decreased it. The optogenetic inhibition of VLS D2-MSN activity increased the formalin-induced pain-related behavior in mice, thus suggesting the inhibitory effect of VLS D2-MSN activity on chemical nociceptive behavior, in contrast to previous reports that the VMS D2-MSNs could not involve the behavior. Therefore, the VLS D2-MSNs exhibited region-specific roles in nociception.

An inhibitory circuit from central amygdala to zona incerta drives pain-related behaviors in mice.

Central amygdala neurons expressing protein kinase C-delta (CeA-PKCδ) are sensitized following nerve injury and promote pain-related responses in mice. The neural circuits underlying modulation of pain-related behaviors by CeA-PKCδ neurons, however, remain unknown. In this study, we identified a neural circuit that originates in CeA-PKCδ neurons and terminates in the ventral region of the zona incerta (ZI), a subthalamic structure previously linked to pain processing. Behavioral experiments show that chemogenetic inhibition of GABAergic ZI neurons induced bilateral hypersensitivity in uninjured mice and contralateral hypersensitivity after nerve injury. In contrast, chemogenetic activation of GABAergic ZI neurons reversed nerve injury-induced hypersensitivity. Optogenetic manipulations of CeA-PKCδ axonal terminals in the ZI further showed that inhibition of this pathway reduces nerve injury-induced hypersensitivity whereas activation of the pathway produces hypersensitivity in the uninjured paws. Altogether, our results identify a novel nociceptive inhibitory efferent pathway from CeA-PKCδ neurons to the ZI that bidirectionally modulates pain-related behaviors in mice.

The possibility of alleviating chronic neuropathic pain and related behaviors by the direct suppression of the parabrachial nucleus

ObjectiveThis study aims to observe the effects of direct suppression of the parabrachial nucleus (PBN) on chronic neuropathic pain (CNP) and CNP-related behaviors in mice. MethodsA CNP model was established using partial sciatic nerve ligation (PSNL) in mice. Two groups were established: the experimental (PSNL) group and the control (sham) group. An assessment of PBN-region c-Fos expression was conducted following von Frey hair stimulation in the PSNL group and the sham group, and the effects of pain induction were detected using behavioral experiments. The PBN activity of the mice with CNP was manipulated using the designer receptors exclusively activated by designer drugs method. Effective and empty virus groups were used to study the effects of PBN activity inhibition on the pain threshold and pain-related behavior in mice with CNP. ResultsThe mechanical pain threshold (MPT) of the mice in the PSNL group was significantly lower than in the sham group. After von Frey stimulation, the c-Fos-positive, PBN-region neurons in the PSNL group were increased compared with the sham group. The central distance in the open field test and the time spent in the central area were lower in the PSNL group than in the sham group. The mice in the PSNL group had a lower duration and fewer entries in the open arm of the elevated plus-maze than the mice in the sham group. There was no difference in immobility time between the PSNL group and the sham group. PBN activity inhibition in mice with CNP did not affect their MPT or anxiety-like behavior. ConclusionCNP can induce anxiety-like behavior and increase PBN-induced pain in mice. However, direct inhibition of the PBN neuron activity alone cannot improve CNP or CNP-related behavior.

EXTENSIVE POLYGENIC OVERLAP BETWEEN SCHIZOPHRENIA AND CORTICAL BRAIN STRUCTURE

Early-life exposure to Δ9-tetrahydrocannabinol (Δ9-THC), the intoxicating constituent of cannabis, may produce enduring neurochemical changes in brain structures involved in the regulation of sociality but it is still unclear how such changes influence social behavior later in life. In the present study, we exposed male mice to moderate daily doses of Δ9-THC (5 mg/kg, intraperitoneal) during adolescence (postnatal day, PND, 30 to 43) and, when animals reached adulthood (PND70), we assessed their performance in the three-chamber social interaction task before and 3 weeks after injection of the chemical irritant formalin (1 % vol, intraplantar), which produces both immediate and persistent pain-related behaviors in mice. Prior Δ9-THC treatment did not alter social interaction in control adult mice but disrupted it in animals that developed lasting sensory abnormalities following formalin injection. The findings suggest that frequent exposure to Δ9-THC during adolescence causes in male mice a dormant dysfunction in social behavior which can be unmasked in adulthood when the animals experience an aversive state.

Investigating the Role of Ly6G+ Neutrophils in Incisional and Inflammatory Pain by Multidimensional Pain-Related Behavioral Assessments: Bridging the Translational Gap

In recent years, preclinical pain research has failed to develop genuinely new analgesics for clinical use. This fact is reflected by a high number of patients, limited drug efficacy accompanied by side effects, and a long-term opioid intake. Two main aspects have been addressed, which hinder translation: the use of non-relevant pain models and a mismatch between pain-related outcomes in preclinical and clinical studies. Conversely, disease-specific pain models that mirror more closely the clinical situation and multidimensional behavioral outcome measures that objectively and reproducibly assess relevant pain-related symptoms in a preclinical setting could improve translation. Mechanistically, a matter of debate is the role of Ly6G+ neutrophil granulocytes (NGs) for pain. NGs are essential to eliminate pathogens and promote the wound healing process. For this purpose, there is a need to release various pro- and anti-inflammatory mediators, some of which could ameliorate or enhance pain. However, the contribution of NGs to different pain entities is contradictory for reflex-based tests, and completely unknown in the context of non-evoked pain (NEP) and movement-evoked pain (MEP). First, we combined withdrawal reflex-based assays with novel video-based assessments for NEP- and MEP-related behavior in two mouse pain models. The pain models utilized in this study were incision (INC) and pathogen/adjuvant-induced inflammation (CFA), translating well to postsurgical and inflammatory pain entities. Second, we depleted NGs and applied a set of behavioral assessments to investigate the role of NG migration in different pain modalities. Our comprehensive behavioral approach identified pain-related behaviors in mice that resemble (NEP) or differentiate (MEP) behavioral trajectories in comparison to mechanical and heat hypersensitivity, thereby indicating modality-dependent mechanisms. Further, we show that injury-induced accumulation of NGs minimally affects pain-related behaviors in both pain models. In conclusion, we report a novel assessment to detect NEP in mice after unilateral injuries using a more unbiased approach. Additionally, we are capable of detecting an antalgic gait for both pain entities with unique trajectories. The different trajectories between MEP and other pain modalities suggest that the underlying mechanisms differ. We further conclude that NGs play a subordinate role in pain-related behaviors in incisional and inflammatory pain.

Frequent Δ9- tetrahydrocannabinol exposure during adolescence impairs sociability in adult mice exposed to an aversive painful stimulus

Early-life exposure to Δ9-tetrahydrocannabinol (Δ9-THC), the intoxicating constituent of cannabis, may produce enduring neurochemical changes in brain structures involved in the regulation of sociality but it is still unclear how such changes influence social behavior later in life. In the present study, we exposed male mice to moderate daily doses of Δ9-THC (5 mg/kg, intraperitoneal) during adolescence (postnatal day, PND, 30 to 43) and, when animals reached adulthood (PND70), we assessed their performance in the three-chamber social interaction task before and 3 weeks after injection of the chemical irritant formalin (1 % vol, intraplantar), which produces both immediate and persistent pain-related behaviors in mice. Prior Δ9-THC treatment did not alter social interaction in control adult mice but disrupted it in animals that developed lasting sensory abnormalities following formalin injection. The findings suggest that frequent exposure to Δ9-THC during adolescence causes in male mice a dormant dysfunction in social behavior which can be unmasked in adulthood when the animals experience an aversive state.

Inhibition of transient receptor potential vanilloid 1 and transient receptor potential ankyrin 1 by mosquito and mouse saliva.

Arthropods are the largest group of living organisms, and among them, mosquitoes spread parasites and viruses causing deadly diseases. They can easily spread these pathogens because of their painless skin piercing. Although the lack of pain is mainly due to the thinness of their fascicle, it is possible that mosquito saliva, which is discharged during their piercing, might also contribute to it. If mosquito saliva contains antinociceptive substances, it should act on the sensory neurons innervating the epidermis where there are several ion channels that can detect noxious stimuli, such as the transient receptor potential (TRP) channels. We found that mosquito head homogenates and mouse saliva inhibit TRP vanilloid 1 (TRPV1) and TRP ankyrin 1 (TRPA1) channels, either heterologously expressed in HEK293T cells or endogenously expressed in native mouse sensory neurons. Among the different substances contained in mosquito head homogenates or mouse saliva, we have also identified sialorphin as a candidate antinociceptive peptide because it showed similar inhibition effects on TRPV1 and TRPA1. Finally, we confirmed the antinociceptive effects of mosquito head homogenates, mouse saliva, and sialorphin in vivo by observing decreased pain-related behaviors in mice coinjected with these substances. Similar inhibitory effects of mosquito head homogenates and mouse saliva on TRPV1 and TRPA1 suggest that the antinociceptive effects of saliva are universal, which could explain why many animals including humans often lick their wounds. These findings would lead to the development of novel and safe antinociceptive agents.

Dual and Opposing Functions of the Central Amygdala in the Modulation of Pain

SUMMARYPain perception is essential for survival and can be amplified or suppressed by expectations, experiences, and context. The neural mechanisms underlying bidirectional modulation of pain remain largely unknown. Here, we demonstrate that the central nucleus of the amygdala (CeA) functions as a pain rheostat, decreasing or increasing pain-related behaviors in mice. This dual and opposing function of the CeA is encoded by opposing changes in the excitability of two distinct subpopulations of GABAergic neurons that receive excitatory inputs from the parabrachial nucleus (PB). Thus, cells expressing protein kinase C-delta (CeA-PKCδ) are sensitized by nerve injury and increase pain-related responses. In contrast, cells expressing somatostatin (CeA-Som) are inhibited by nerve injury and their activity drives antinociception. Together, these results demonstrate that the CeA can amplify or suppress pain in a cell-type-specific manner, uncovering a previously unknown mechanism underlying bidirectional control of pain in the brain.

Chemokines CCL2 and CCL7, but not CCL12, play a significant role in the development of pain-related behavior and opioid-induced analgesia

The complex neuroimmunological interactions mediated by chemokines are suggested to be responsible for the development of neuropathic pain. The lack of knowledge regarding the detailed pathomechanism of neuropathy is one reason for the lack of optimally efficient therapies. Recently, several lines of evidence indicated that expression of CCR2 is increased in spinal cord neurons and microglial cells after peripheral nerve injury. It was previously shown that administration of CCR2 antagonists induces analgesic effects; however, the role of CCR2 ligands in neuropathic pain still needs to be explained. Thus, the goal of our studies was to investigate the roles of CCL2, CCL7, and CCL12 in neuropathic pain development and opioid effectiveness. The experiments were conducted on primary glial cell cultures and two groups of mice: naive and neuropathic. We used chronic constriction injury (CCI) of the sciatic nerve as a neuropathic pain model. Mice intrathecally received chemokines (CCL2, CCL7, CCL12) at a dose of 10, 100 or 500 ng, neutralizing antibodies (anti-CCL2, anti-CCL7) at a dose of 1, 4 or 8 μg, and opioids (morphine, buprenorphine) at a dose of 1 μg. The pain-related behaviors were assessed using the von Frey and cold plate tests. The biochemical analysis of mRNA expression of glial markers, CCL2, CCL7 and CCL12 was performed using quantitative reverse transcriptase real-time PCR. We demonstrated that CCI of the sciatic nerve elevated spinal expression of CCL2, CCL7 and CCL12 in mice, in parallel with microglia and astroglial activation markers. Moreover, intrathecal injection of CCL2 and CCL7 induced pain-related behavior in naive mice in a dose-dependent manner. Surprisingly, intrathecal injection of CCL12 did not influence nociceptive transmission in naive or neuropathic mice. Additionally, we showed for the first time that intrathecal injection of CCL2 and CCL7 neutralizing antibodies not only attenuated CCI-induced pain-related behaviors in mice but also augmented the analgesia induced by morphine and buprenorphine. In vitro studies suggest that both microglia and astrocytes are an important cellular sources of the examined chemokines. Our results revealed the crucial roles of CCL2 and CCL7, but not CCL12, in neuropathic pain development and indicated that pharmacological modulation of these factors may serve as a potential therapeutic target for new (co)analgesics.

PC-FACS

PC-FACS

The Amelioration of Pain-Related Behavior in Mice with Chronic Spinal Cord Injury Treated with Neural Stem/Progenitor Cell Transplantation Combined with Treadmill Training.

Progress in regenerative medicine is realizing the possibility of neural regeneration and functional recovery in spinal cord injury (SCI). Recently, rehabilitation has attracted much attention with respect to the synergistic promotion of functional recovery in combination with neural stem/progenitor cell (NS/PC) transplantation, even in the chronic refractory phase of SCI. Nevertheless, sensory disturbance is one of the most prominent sequelae, even though the effects of combination or single therapies have been investigated mostly in the context of motor recovery. To determine how combination therapy with treadmill training (TMT) and NS/PC transplantation affects the manifestation of thermal allodynia and tactile hyperalgesia in chronic phase SCI, four groups of SCI mice were used to assess pain-related behavior and histological changes: combined transplantation and TMT therapy, transplantation only, TMT only, and control groups. Thermal allodynia and coarse touch-pressure hyperalgesia exhibited significant recovery in the combined therapy group in comparison with controls, whereas there were no significant differences with fine touch-pressure hyperalgesia and motor function. Further investigation revealed fewer fibers remaining in the posterior funiculus, which contained the tracts associated with the two modalities showing less recovery; that is, touch-pressure hyperalgesia and motor function. A significant correlation was only observed between these two modalities. Although no remarkable histological recovery was found within the lesion epicenter, changes indicating amelioration of pain were observed in the lumbar enlargement of the combination therapy group. Our results suggest that amelioration of thermal allodynia and tactile hyperalgesia can be brought about by the additive effect of NS/PC transplantation and TMT. The degree of recovery seems dependent on the distribution of damage.

Neonatal Sleep Restriction Increases Nociceptive Sensitivity in Adolescent Mice

Sleep loss in infants may have a negative effect on the functional and structural development of the nociceptive system. We tested the hypothesis that neonatal sleep restriction induces a long-term increase of pain-related behaviors in mice and that this hypersensitivity occurs due to changes in the neuronal activity of nociceptive pathways. We aim to investigate the effects of sleep loss in neonatal mice on pain behaviors of adolescent and adult mice in a sex-dependent manner. We also analyzed neuroanatomical and functional changes in pain pathways associated with behavioral changes. An experimental animal study. A basic sleep research laboratory at Universidade Federal de São Paulo in Brazil. Neonatal mice at postnatal day (PND) 12 were randomly assigned to either control (CTRL), maternal separation (MS), or sleep restriction (SR) groups. MS and SR were performed 2 hours a day for 10 days (PND 12 until PND 21). The gentle handling method was used to prevent sleep. At PND 21, PND 35, or PND 90, the mice were tested for pain-related behaviors. Their brains were harvested and immunohistochemically stained for c-Fos protein in the anterior cingulate cortex, primary somatosensory cortex, and periaqueductal gray (PAG). Neonatal SR significantly increased nociceptive sensitivity in the hot plate test in adolescent mice (-23.5% of pain threshold). This alteration in nociceptive response was accompanied by a decrease in c-Fos expression in PAG (-40% of c-Fos positive cells compared to the CTRL group). The hypersensitivity found in adolescent mice was not present in adult animals, and all mice showed a comparable nociceptive response. Even using a mild manipulation method, in which a minimal amount of handling was applied to maintain wakefulness, sleep deprivation was a stressful event evidenced by higher corticosterone levels. Repeated exposures to sleep loss during early life were able to induce changes in the nociceptive response associated with alterations in neural activity in descending control of pain. Brain maturation, hypersensitivity, neuronal activity, nociception, pain, periaqueductal gray, postnatal development, sleep, sleep deprivation.

Involvement of TRPV1-ANO1 Interactions in Pain-Enhancing Mechanisms.

Primary sensory neurons detect potentially dangerous environmental situations via many "sensor" proteins located on the plasma membrane. Although receptor-type cation channels are thought to be the major sensors in sensory neurons, anion channels are also important players in the peripheral nervous system. Recently, we showed that transient receptor potential vanilloid 1 (TRPV1) interacts with anoctamin 1 (ANO1, also called TMEM16A) in primary sensory neurons and that this interaction enhanced TRPV1-mediated pain sensation. In that study, we induced ANO1 currents by application of capsaicin to small DRG neurons and showed that ANO1-dependent depolarization following TRPV1 activation could evoke more action potentials. Furthermore, capsaicin-evoked pain-related behaviors in mice were strongly inhibited by a selective ANO1 blocker. Together these findings indicate that selective ANO1 inhibition can reduce pain sensation. We also investigated non-specific inhibitory effects on ion channel activities to control ion dynamics via the TRPV1-ANO1 complex. We found that 4-isopropylcyclohexanol (4-iPr-CyH-OH) had an analgesic effect on burning pain sensations through its inhibition of TRPV1 and ANO1 together. Additionally, 4-iPr-CyH-OH did not have clear agonistic effects on TRPV1, TRPA1, and ANO1 activity individually. These results indicate that 4-iPr-CyH-OH could function globally to mediate TRP-ANO1 complex functions to reduce skin hypersensitivity and could form the basis for novel analgesic agents.

Pharmacological validation of voluntary gait and mechanical sensitivity assays associated with inflammatory and neuropathic pain in mice

The urgent need for more effective analgesic treatment options has prompted a re-evaluation of the behavioral tests used to assess pain in pre-clinical research, with an emphasis on inclusion of more voluntary, un-evoked behavioral assessments of pain. In order to validate voluntary gait analysis and a voluntary mechanical conflict-avoidance assay, we tested mouse models of neuropathy (spared nerve injury) and inflammation (complete Freund's adjuvant) alongside reflexive measures of mechanical and thermal hypersensitivity. To establish whether the observed changes in behavioral responses were pain-related, known analgesics (buprenorphine, gabapentin, carprofen) were also administered. Spared nerve injury persistently altered several gait indices, whereas complete Freund's adjuvant caused only transient changes. Furthermore, known analgesics could not reverse these gait changes, despite demonstrating their previously established efficacy in reflexive measures of mechanical and thermal hypersensitivity. In contrast, the mechanical conflict-avoidance assay demonstrated aversion in mice with neuropathy and inflammation-induced hypersensitivity, which could both be reversed by analgesics. We conclude that voluntary gait changes in rodent neuropathic and inflammatory pain models are not necessarily indicative of pain-related adaptations. On the other hand, mechanical conflict-avoidance represents a valid operant assay for quantifying pain-related behaviors in mice that can be reversed by known analgesics.

Therapeutic potential for leukocyte elastase in chronic pain states harboring a neuropathic component

Neuropathic pain is an integral component of several chronic pain conditions and poses a major health problem worldwide. Despite emerging understanding of mechanisms behind neuropathic pain, the available treatment options are still limited in efficacy or associated with side effects, therefore making it necessary to find viable alternatives. In a genetic screen, we recently identified SerpinA3N, a serine protease inhibitor secreted in response to nerve damage by the dorsal root ganglion neurons and we showed that SerpinA3N acts against induction of neuropathic pain by inhibiting the T-cell- and neutrophil-derived protease, leucocyte elastase (LE). In the current study, via detailed in vivo pharmacology combined with analyses of evoked- and spontaneous pain-related behaviors in mice, we report that on systemic delivery, a single dose of 3 independent LE inhibitors can block established nociceptive hypersensitivity in early and late phases in the spared nerve injury model of traumatic neuropathic pain in mice. We further report the strong efficacy of systemic LE inhibitors in reversing ongoing pain in 2 other clinically relevant mouse models-painful diabetic neuropathy and cancer pain. Detailed immunohistochemical analyses on the peripheral tissue samples revealed that both T-Lymphocytes and neutrophils are the sources of LE on peripheral nerve injury, whereas neutrophils are the primary source of LE in diabetic neuropathic conditions. In summary, our results provide compelling evidence for a strong therapeutic potential of generic LE inhibitors for the treatment of neuropathic pain and other chronic pain conditions harboring a neuropathic pain component.

Nonviral vector plasmid DNA encoding human proenkephalin gene attenuates inflammatory and neuropathic pain-related behaviors in mice

Inflammatory pain and neuropathic pain are major clinical health issues that represent considerable social and economic burden worldwide. In the present study, we investigated the anti-nociceptive efficacy of delivery of human proenkephalin gene by a plasmid DNA vector (pVAX1-PENK) on complete Freund’s adjuvant (CFA) induced inflammatory pain and spared nerve injury (SNI) induced neuropathic pain in mice. Mice were intramuscularly or intrathecally administered pVAX1 or pVAX1-PENK, respectively. Pain thresholds in the pVAX1-PENK treated mice were significantly higher at day 3, then reached a peak at day 7 and lasted until day 28 after gene transfer, and the analgesic effect of pVAX1-PENK was blocked with naloxone hydrochloride. In contrast, pVAX1 treated mice did not significantly improve pain thresholds. These results indicate that peripheral or spinal delivery of a plasmid encoding human proenkephalin gene provides a potential therapeutic strategy for inflammatory pain and neuropathic pain.

Pain-enhancing mechanism through interaction between TRPV1 and anoctamin 1 in sensory neurons

The capsaicin receptor transient receptor potential cation channel vanilloid 1 (TRPV1) is activated by various noxious stimuli, and the stimuli are converted into electrical signals in primary sensory neurons. It is believed that cation influx through TRPV1 causes depolarization, leading to the activation of voltage-gated sodium channels, followed by the generation of action potential. Here we report that the capsaicin-evoked action potential could be induced by two components: a cation influx-mediated depolarization caused by TRPV1 activation and a subsequent anion efflux-mediated depolarization via activation of anoctamin 1 (ANO1), a calcium-activated chloride channel, resulting from the entry of calcium through TRPV1. The interaction between TRPV1 and ANO1 is based on their physical binding. Capsaicin activated the chloride currents in an extracellular calcium-dependent manner in HEK293T cells expressing TRPV1 and ANO1. Similarly, in mouse dorsal root ganglion neurons, capsaicin-activated inward currents were inhibited significantly by a specific ANO1 antagonist, T16Ainh-A01 (A01), in the presence of a high concentration of EGTA but not in the presence of BAPTA [1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid]. The generation of a capsaicin-evoked action potential also was inhibited by A01. Furthermore, pain-related behaviors in mice treated with capsaicin, but not with αβ-methylene ATP, were reduced significantly by the concomitant administration of A01. These results indicate that TRPV1-ANO1 interaction is a significant pain-enhancing mechanism in the peripheral nervous system.

A pore forming peptide from spider Lachesana sp. venom induced neuronal depolarization and pain

BackgroundArachnoid venoms contain numerous peptides with ion channel modifying and cytolytic activities. MethodsWe developed a green fluorescent protein (GFP)-based assay that can monitor the changes in currents through overexpressed inwardly rectifying K+ channels (Kir2.1), in which GFP expression was increased by blockade of Kir2.1 current. Using this assay, we screened venom of many spider species. A peptide causing GFP decreasing effect was purified and sequenced. Electrophysiological and pain-inducing effects of the peptide were analyzed with whole-cell patch-clamp recordings and hot-plate test, respectively. ResultsAmong venoms we screened, soluble venom from Lachesana sp. decreased the GFP expression. Purification and sequencing of the peptide showed that the peptide is identical to a pore-forming peptide purified from Lachesana tarabaevi venom. Whole cell patch-clamp recordings revealed that the peptide had no effect on Kir2.1 current. Instead, it induced a current that was attributable to the pore-formation of the peptide. The peptide was selectively incorporated into hyperpolarized, i.e., Kir2.1 expressing, cells and for this reason the peptide decreased GFP expression in our Kir2.1 assay. The pore-formation positively shifted the reversal potential and induced burst firings in the hippocampal neurons in a synaptic current-independent way. The application of the Lachesana sp. peptide induced pain-related behavior in mice. ConclusionsThe peptide, which was found in Lachesana sp. venom, formed pores and thereby depolarized neurons and induced pain. General significanceOur data suggested an additional physiological role of the pore-forming peptides.

Endocannabinoids decrease neuropathic pain-related behavior in mice through the activation of one or both peripheral CB1 and CB2 receptors

The two most studied endocannabinoids are anandamide (AEA), principally catalyzed by fatty-acid amide hydrolase (FAAH), and 2-arachidonoyl glycerol (2-AG), mainly hydrolyzed by monoacylglycerol lipase (MGL). Inhibitors targeting these two enzymes have been described, including URB597 and URB602, respectively. Several recent studies examining the contribution of CB1 and/or CB2 receptors on the peripheral antinociceptive effects of AEA, 2-AG, URB597 and URB602 in neuropathic pain conditions using either pharmacological tools or transgenic mice separately have been reported, but the exact mechanism is still uncertain. Mechanical allodynia and thermal hyperalgesia were evaluated in 436 male C57BL/6, cnr1KO and cnr2KO mice in the presence or absence of cannabinoid CB1 (AM251) or CB2 (AM630) receptor antagonists in a mouse model of neuropathic pain. Peripheral subcutaneous injections of AEA, 2-AG, WIN55,212-2 (WIN; a CB1/CB2 synthetic agonist), URB597 and URB602 significantly decreased mechanical allodynia and thermal hyperalgesia. These effects were inhibited by both cannabinoid antagonists AM251 and AM630 for treatments with 2-AG, WIN and URB602 but only by AM251 for treatments with AEA and URB597 in C57BL/6 mice. Furthermore, the antinociceptive effects for AEA and URB597 were observed in cnr2KO mice but absent in cnr1KO mice, whereas the effects of 2-AG, WIN and URB602 were altered in both of these transgenic mice. Complementary genetic and pharmacological approaches revealed that the anti-hyperalgesic effects of 2-AG and URB602 required both CB1 and CB2 receptors, but only CB2 receptors mediated its anti-allodynic actions. The antinociceptive properties of AEA and URB597 were mediated only by CB1 receptors.