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Neuropathic pain (NP) remains an untreatable disease due to the complex pathophysiology that involves the whole pain neuraxis including the forebrain. Sensory dysfunctions such as allodynia and hyperalgesia are only part of the symptoms associated with neuropathic pain that extend to memory and affectivity deficits. The development of multi-target molecules might be a promising therapeutic strategy against the symptoms associated with NP. 2-pentadecyl-2-oxazoline (PEA-OXA) is a plant-derived agent, which has shown effectiveness against chronic pain and associated neuropsychiatric disorders. The molecular mechanisms by which PEA-OXA exerts its effects are, however, only partially known. In the current study, we show that PEA-OXA, besides being an alpha2 adrenergic receptor antagonist, also acts as a modulator at histamine H3 receptors, and report data on its effects on sensory, affective and cognitive symptoms associated with the spared nerve injury (SNI) model of neuropathic pain in mice. Treatment for 14 days with PEA-OXA after the onset of the symptoms associated with neuropathic pain resulted in the following effects: (i) allodynia was decreased; (ii) affective/cognitive impairment associated with SNI (depression, spatial, and working memories) was counteracted; (iii) long-term potentiation in vivo in the lateral entorhinal cortex-dentate gyrus (perforant pathway, LPP) was ameliorated, (iv) hippocampal glutamate, GABA, histamine, norepinephrine and dopamine altered levels after peripheral nerve injury were reversed, (v) expression level of the TH positive neurons in the Locus Coeruleus were normalized. Thus, a 16-day treatment with PEA-OXA alleviates the sensory, emotional, cognitive, electrophysiological and neurochemical alterations associated with SNI-induced neuropathic pain.

The study investigated the role of the metabotropic glutamate receptor subtype 7 (mGluR7) in pain signalling in the dorsal striatum of sham and neuropathic rats. Supraspinal circuitries involved in the dorsal striatum control of pain were also explored. In the sham rats, microinjection of N,N'-bis(diphenylmethyl)-1,2-ethanediamine (AMN082), a selective mGluR7 positive allosteric modulator, into the dorsal striatum, facilitated pain, increased the activity of the ON cells and inhibited the activity of the OFF cells in the rostral ventromedial medulla, and decreased glutamate levels in the dorsal striatum. Conversely, AMN082 inhibited pain and the activity of the ON cells while increased the activity of the OFF cells in rats with spared nerve injury (SNI) of the sciatic nerve. AMN082 also decreased glutamate levels in the dorsal striatum of SNI rats. The effect of AMN082 on mechanical allodynia and glutamate release was blocked by 6-(2,4-dimethylphenyl)-2-ethyl-6,7-dihydro-4(5H)-benzoxazolone (ADX71743), a selective mGluR7 negative allosteric modulator. Moreover, in the sham rats, AMN082 increased the activity of total nociceptive convergent neurons in the dorsal reticular nucleus while in the SNI rats, such activity was decreased. The administration of lidocaine into the subthalamic nucleus abolished the effect of AMN082 on the total nociceptive convergent neurons in the sham rats but not in the SNI rats. Thus, the dual effect of mGluR7 in facilitating or inhibiting pain responses may be due to the recruitment of different pathways of the basal ganglia, the indirect or direct pathway, in physiological or pathological conditions, respectively.

Inflammation plays a key role in the pathogenesis of several chronic diseases. The urokinase plasminogen activator receptor (uPAR) exerts a plethora of functions in both physiological and pathological processes, including inflammation. In this study, we evaluated the anti-inflammatory effect of a novel peptide ligand of uPAR, UPARANT, in different animal models of inflammation. Rats and mice were divided in different groups (n = 5) for single or repeated administration of vehicle (9% DMSO in 0.9% NaCl), UPARANT (6, 12 and 24 mg/kg) or dexamethasone (2 mg/kg). Animals were subjected to carrageenan-induced paw oedema or zymosan-induced peritonitis. UPARANT effects were tested on: (1) the carrageenan-induced paw oedema volume, (2) the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) and the nitrite/nitrate (NOx) levels in the paw exudates, (3) cells recruitment into the peritoneal cavity after zymosan injection and (4) NOx levels in the peritoneal lavage. UPARANT (12 and 24 mg/kg) reduced inflammation in both experimental paradigms. Analysis of pro-inflammatory enzymes revealed that administration of UPARANT reduced iNOS, COX2 and NO over-production. Our study provides a solid evidence that UPARANT reduces the severity of inflammation in diverse animal models, thus representing a novel anti-inflammatory drug with potential advantages with respect to the typical steroidal agents.

The mechanisms underlying neuropathic pain are poorly understood. Here we show the unexplored role of the hydroxyl carboxylic acid receptor type 2 (HCAR2) in 2 models of neuropathic pain. We used an oral treatment with dimethyl fumarate and the HCAR2 endogenous ligand β-hydroxybutyrate (BHB) in wild-type (WT) and HCAR2-null mice. We found an up-regulation of the HCAR2 in the sciatic nerve and the dorsal root ganglia in neuropathic mice. Accordingly, acute and chronic treatment with dimethylfumarate (DMF) and BHB reduced the tactile allodynia. This effect was completely lost in the HCAR2-null mice after a 2-d starvation protocol, in which the BHB reached the concentration able to activate the HCAR2-reduced tactile allodynia in female WT mice, but not in the HCAR2-null mice. Finally, we showed that chronic treatment with DMF reduced the firing of the ON cells (cells responding with an excitation after noxious stimulation) of the rostral ventromedial medulla. Our results pave the way for investigating the mechanisms by which HCAR2 regulates neuropathic pain plasticity.-Boccella, S., Guida, F., De Logu, F., De Gregorio, D., Mazzitelli, M., Belardo, C., Iannotta, M., Serra, N., Nassini, R., de Novellis, V., Geppetti, P., Maione, S., Luongo, L. Ketones and pain: unexplored role of hydroxyl carboxylic acid receptor type 2 in the pathophysiology of neuropathic pain.

D-Aspartate (D-Asp) is a free D-amino acid detected in multiple brain regions and putative precursor of endogenous N-methyl-D-aspartate (NMDA) acting as agonist at NMDA receptors. In this study, we investigated whether D-Asp (20 mM) in drinking solution for 1 month affects pain responses and pain-related emotional, and cognitive behaviour in a model of neuropathic pain induced by the spared nerve injury (SNI) of the sciatic nerve in mice. SNI mice developed mechanical allodynia and motor coordination impairment 30 days after SNI surgery. SNI mice showed cognitive impairment, anxiety and depression-like behaviour, reduced sociability in the three chamber sociability paradigm, increased expression of NR2B subunit of NMDA receptor and Homer 1a in the medial prefrontal cortex (mPFC). The expression of (post synaptic density) PSD-95 and Shank 1was instead unaffected in the mPFC of the SNI mice. Treatment with D-Asp drinking solution, started right after the SNI (day 0), alleviated mechanical allodynia, improved cognition and motor coordination and increased social interaction. D-Asp also restored the levels of extracellular D-Asp, Homer 1a and NR2B subunit of the NMDA receptor to physiological levels and reduced Shank1 and PSD-95 protein levels in the mPFC. Amitriptyline, a tricyclic antidepressant used also to alleviate neuropathic pain in humans, reverted mechanical allodynia and cognitive impairment, and unlike D-Asp, was effective in reducing depression and anxiety-like behaviour in the SNI mice and increased PSD protein level. Altogether these findings demonstrate that D-Asp improves sensorial, motor and cognitive-like symptoms related to chronic pain possibly through glutamate neurotransmission normalization in neuropathic mice.

D-aspartate levels in the brain are regulated by the catabolic enzyme D-aspartate oxidase (DDO). D-aspartate activates NMDA receptors, and influences brain connectivity and behaviors relevant to schizophrenia in animal models. In addition, recent evidence reported a significant reduction of D-aspartate levels in the post-mortem brain of schizophrenia-affected patients, associated to higher DDO activity. In the present work, microdialysis experiments in freely moving mice revealed that exogenously administered D-aspartate efficiently cross the blood brain barrier and stimulates L-glutamate efflux in the prefrontal cortex (PFC). Consistently, D-aspartate was able to evoke L-glutamate release in a preparation of cortical synaptosomes through presynaptic stimulation of NMDA, mGlu5 and AMPA/kainate receptors. In support of a potential therapeutic relevance of D-aspartate metabolism in schizophrenia, in vitro enzymatic assays revealed that the second-generation antipsychotic olanzapine, differently to clozapine, chlorpromazine, haloperidol, bupropion, fluoxetine and amitriptyline, inhibits the human DDO activity. In line with in vitro evidence, chronic systemic administration of olanzapine induces a significant extracellular release of D-aspartate and L-glutamate in the PFC of freely moving mice, which is suppressed in Ddo knockout animals. These results suggest that the second-generation antipsychotic olanzapine, through the inhibition of DDO activity, increases L-glutamate release in the PFC of treated mice.

Depressive symptoms and other neuropsychiatric dysfunctions are common in neurodegenerative disorders, including chronic pain and dementia. A correlation between the β-amyloid protein accumulation and the development of depression has been suggested, however the underlying mechanisms are unknown. d-Aspartate (d-Asp) is a free d-amino acid found in the mammalian brain and involved in neurological and psychiatric processes, such as cognition and affective disorders. In this study we have investigated the effects of a repeated treatment with d-Asp in a long-lasting (12 months) model of neuropathic pain, the spared nerve injury (SNI), in mice. Specifically, we evaluated i) the pain sensitivity and related emotional/cognitive dysfunctions induced by SNI, ii) possible changes in the β-amyloid protein accumulation in specific brain regions involved in pain mechanisms ii) possible changes in steroids level in neuropathic animals with or without d-Asp in the same brain areas. SNI mice showed an increase of the insoluble form of Aβ

In the pathogenesis of neuropathic pain, the conversion of astrocytes in the reactive state and the ras-dependent Erk-mediated pathway play an important role. Zoledronic acid (ZOL) is a potent inhibitor of the latter pathway, but its activity in neurological diseases is hampered by its biodistribution that is almost exclusively limited to the bone. We have developed nanotechnological devices able to increase the accumulation of ZOL in extra bone sites. In this work, we have evaluated the effects of ZOL-encapsulating PEGylated liposomes (LipoZOL) on an animal model of neuropathic pain. We have found that 2 iv administrations (10 μg of ZOL, either as free or encapsulated into liposomes) at days 2 and 4 after the injury markedly reduced mechanical hypersensitivity at 3 and 7 days after nerve injury. On the other hand, free ZOL did not exert any significant alteration of the mechanical threshold. Immunohistochemical analysis of spinal cord revealed that GFAP-labeled astrocytes appeared hypertrophic activated cells in the ispilateral dorsal horn of spinal cord 7 days after SNI. LipoZOL significantly changed astrocyte morphology, by inducing a protective phenotype, without changing the total cell number. Moreover, the astrocytes of the spinal cord of LipoZOL-treated mice were positive for interleukin-10. Delivery of ZOL into the CNS was confirmed by biodistribution of fluorescently labeled liposomes. In particular, liposomes accumulated in the liver and kidney in both groups of normal and neuropathic animals; on the other hand, only in the case of neuropathic animals, a fluorescence increase in the brain and spinal cord occurred only in neuropathic animals at 30 min and 1 h. These data demonstrate that ZOL, only by using a delivery system able to cross the altered BBB, could be a new opportunity to treat neuropathic pain.

This study analyzed the effect of intra-ventrolateral periaqueductal grey (VL PAG) cannabinoid receptor (CB) stimulation on pain responses and rostral ventromedial medulla (RVM) neural activity in the chronic constriction injury (CCI) model of neuropathic pain in rats. Interaction between CB 1 and metabotropic glutamate 1 and 5 (mGlu 1 /mGlu 5 ) receptors was also investigated together with the expression of the CB 1 receptor associated Gai3 and cannabinoid receptor interacting 1a (CRIP 1a) proteins and the endocannabinoid synthesising and hydrolysing enzymes. In rats not subjected to CCI-induced pain, intra-VL PAG (R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl) pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl]-1-naphthalenylmethanone mesylate (WIN 55,212-2) (2-4-8 nmol), a CB receptor agonist, increased the tail flick latency and changed the ongoing activity of RVM OFF and the tail flick-related activity of the ON and OFF cells, accordingly. These effects were prevented by SR141716A and MPEP, selective CB 1 and mGlu 5 receptor antagonists, respectively, though not by CPCCOEt, a selective mGlu 1 receptor antagonist. A higher dose up to 16 nmol of WIN 55,212-2 was necessary to increase tail flick latency and change ON and OFF cell activity in CCI rats. Consistently, CCI rats showed a decrease in the expression of CB 1 receptors, NAPE-PLD, Gαi3 and CRIP 1a proteins;the expression of diacylglycerol lipase A (DAGLA) was increased while fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MGL) did not change. As in control rats, MPEP and SR141716A also blocked WIN 55,212-2- induced effects in CCI rats. These data demonstrate a down regulation of the endocannabinoid system and a functional interaction between mGlu5 and CB 1 receptors for cannabinoid-mediated effect in the PAG-RVM pain circuitry in neuropathic pain inflicted rats.

Autism and autism spectrum disorders (ASDs) are heterogeneous complex neuro-developmental disorders characterized by dysfunctions in social interaction and communication skills. Their pathogenesis has been linked to interactions between genes and environmental factors. Consistent with the evidence of certain similarities between immune cells and neurons, autistic children also show an altered immune response of peripheral blood mononuclear cells (PBMCs). In this study, we investigated the activation of caspases, cysteinyl aspartate-specific proteases involved in apoptosis and several other cell functions in PBMCs from 15 ASD children compared to age-matched normal healthy developing controls. The mRNA levels for caspase-1, -2, -4, -5 were significantly increased in ASD children as compared to healthy subjects. Protein levels of Caspase-3, -7, -12 were also increased in ASD patients. Our data are suggestive of a possible role of the caspase pathway in ASD clinical outcome and of the use of caspase as potential diagnostic and/or therapeutic tools in ASD management.

N-acyl-vanillamide (NAVAM) analogues of the natural pungent principle of capsicum, capsaicin, were developed several years ago as potential non-pungent analgesic compounds. N-oleoyl-vanillamide (olvanil) and N-arachidonoy-vanillamide (arvanil), in particular, were described in several publications and patents to behave as potent anti-hyperalgesic compounds in experimental models of chronic and inflammatory pain, and to activate both "capsaicin receptors", i.e. the transient receptor potential of vanilloid type-1 (TRPV1) channel, and, either directly or indirectly, cannabinoid receptors of type-1. Here we report the biochemical and pharmacological characterization of a so far neglected NAVAM, N-palmitoyl-vanillamide (palvanil), and propose its possible use instead of capsaicin, as a possible topical analgesic. Palvanil exhibited a kinetics of activation of human recombinant TRPV1-mediated intracellular calcium elevation significantly slower than that of capsaicin (t(1/2) = 21 s and 8 s, respectively at 1 mu M). Slow kinetics of TRPV1 agonists were previously found to be associated with stronger potencies as TRPV1 desensitizing agents, which in turn are usually associated with lower pungency and stronger anti-hyperalgesic activity. Accordingly, palvanil desensitized the human recombinant TRPV1 to the effect of capsaicin (10 nM) with significantly higher potency than capsaicin (IC(50) = 0.8 nM and 3.8 nM, respectively), this effect reaching its maximum more rapidly (50 and 250 min, respectively). Palvanil was also more potent than capsaicin at desensitizing the stimulatory effect of TRPV1 by low pH together with anandamide, which mimics conditions occurring during inflammation. In the eye-wiping assay carried out in mice, palvanil was not pungent and instead caused a strong and long-lasting inhibition of capsaicin-induced eye-wiping. Finally, intraplantar palvanil inhibited the second phase of the nociceptive response to formalin in mice. In conclusion, palvanil appears to be a non-pungent analogue of capsaicin with stronger desensitizing effects on TRPV1 and hence potentially higher anti-hyperalgesic activity.

The amygdala is a crucial area in controlling the threshold of pain and its emotional component. The present study has evaluated the effect of a metabotropic glutamate 8 receptor (mGluR8) stimulation in the central nucleus of the amygdala (CeA) on the thermoceptive threshold and on CeA serotonin (5-HT), glutamate (Glu), and GABA release in normal and carrageenan-induced inflammatory pain conditions in rats. Furthermore, the activity of rostral ventromedial medulla (RVM) putative “pronociceptive” ON and “antinociceptive” OFF cells has been evaluated. (S)-3,4-Dicarboxyphenylglycine [(S)-3,4-DCPG], a selective mGluR8 agonist, administered into the CeA, did not change 5-HT, Glu, and GABA release, or the thermoceptive threshold, nor did it modify the activity of ON and OFF cells of the RVM in normal animals. In rats treated with carrageenan, intra-CeA (S)-3,4-DCPG perfusion produced antinociception, and increased 5-HT and Glu, whereas it decreased GABA release. Intra-CeA (S)-3,4-DCPG inhibited ON and increased OFF cell activities. Furthermore, an increase inmGluR8gene, protein, and staining, the latter being associated with vesicular GABA transporter-positive profiles, has been found in the CeA after carrageenan-induced inflammatory pain. These results show that stimulation of mGluR8, which was overexpressed within the CeA in inflammatory pain conditions, inhibits nociceptive behavior. Such an effect is associated with an increase in 5-HT and Glu release, a decrease in GABA, and the inhibition of ON- and the stimulation of OFF-cell activities within RVM.

The aim of this study was to obtain evidences of a possible analgesic role for palmitoylethanolamide (PEA) in chronic granulomatous inflammation sustained by mast cell (MC) activation in rats at 96 hours. PEA (200-400-800 μg/mL), locally administered at time 0, reduced in a concentration-dependent manner the expression and release of NGF in comparison with saline-treated controls. PEA prevented nerve formation and sprouting, as shown by histological analysis, reduced mechanical allodynia, evaluated by Von Frey filaments, and inhibited dorsal root ganglia activation. These results were supported by the evidence that MCs in granuloma were mainly degranulated and closely localized near nerve fibres and PEA significantly reduced MC degranulation and nerves fibre formation. These findings are the first evidence that PEA, by the modulation of MC activation, controls pain perception in an animal model of chronic inflammation, suggesting its potential use for the treatment of all those painful conditions in which MC activation is an initial key step.

This article describes three cases of Datura stramonium intake on two nonconsecutive days. In the first case, the patient took a small amount of D. stramonium seeds without showing any symptoms of intoxication. The other two patients had taken a considerable amount of seeds and reported a sudden surge in strength and energy, with some aggressive compulsion towards their peers. They showed delirium as well as confusion and disorientation. The absence of any specific legislation makes D. stramonium a tempting alternative to other psychoactive substances. Thus, it is extremely important to be able to recognize its symptoms so as to be able to diagnose any signs of intoxication properly.

Neuropathic pain is a very complex disease, involving several molecular pathways. Current available drugs are usually not acting on the several mechanisms underlying the generation and propagation of pain. We used spared nerve injury model of neuropathic pain to assess the possible use of human mesenchymal stem cells (hMSCs) as anti-neuropathic tool. Human MSCs were transplanted in the mouse lateral cerebral ventricle. Stem cells injection was performed 4 days after sciatic nerve surgery. Neuropathic mice were monitored 7, 10, 14, 17, and 21 days after surgery. hMSCs were able to reduce pain-like behaviors, such as mechanical allodynia and thermal hyperalgesia, once transplanted in cerebral ventricle. Anti-nociceptive effect was detectable from day 10 after surgery (6 days post cell injection). Human MSCs reduced the mRNA levels of the pro-inflammatory interleukin IL-1beta mouse gene, as well as the neural beta-galactosidase over-activation in prefrontal cortex of SNI mice. Transplanted hMSCs were able to reduce astrocytic and microglial cell activation.

We evaluated the effects of intra-periaqueductal grey (PAG) N-arachidonoyl-serotonin (AA-5-HT), a compound with a "dual" ability to inhibit the fatty acid amide hydrolase (FAAH) and to antagonize transient receptor vanilloid type 1 (TRPV1) receptors, on endocannabinoid levels, rostral ventromedial medulla (RVM) ON and OFF cell activities, thermal nociception (tail flick in anaesthetized rats) and formalin-induced nocifensive responses in awake rats. AA-5-HT increased endocannabinoid levels in the PAG and induced analgesia. Paradoxically, it also depressed the RVM OFF cell, as well as the ON cell activities. The effect of AA-5-HT was mimicked by co-injecting the selective FAAH inhibitor URB597 and the selective TRPV1 antagonist I-RTX into the PAG, which also induced analgesia and inhibition of ON and OFF cell ongoing activities. The recruitment of "alternative" pathways, such as PAG-locus coeruleus (LC)-spinal cord might be responsible for AA-5-HT effect since we found evidence that (i) intra-PAG AA-5-HT increased LC neuron firing activities, and (ii) intrathecal phentolamine or ketanserin prevented the analgesic effect of AA-5-HT. Moreover, intra-PAG AA-5-HT prevented the changes in the ON and OFF cells firing activity induced by intra-paw formalin, and it inverted the formalin-induced increase in LC adrenergic cell activity. All AA-5-HT effects were antagonized by cannabinoid CB1 and TRPV1 receptor antagonists thus suggesting that co-localization of these receptors in the PAG can be an appropriate neural substrate for AA-5-HT-induced analgesia.

Recent studies have shown that activation of the cannabinoid CB(1) receptor by synthetic agonists, and pharmacological elevation of endocannabinoid levels, suppress hyperalgesia and allodynia in animal models of neuropathic pain. However, the concentrations of endocannabinoids in the nervous tissues involved in pain transmission during neuropathic pain have never been measured. Here we have determined the levels of anandamide and 2-arachidonoylglycerol (2-AG), as well as of the analgesic anandamide congener, palmitoylethanolamide (PEA), in three brain areas involved in nociception, i.e. the dorsal raphe (DR), periaqueductal grey (PAG) and rostral ventral medulla (RVM), as well as in the spinal cord (SC), following chronic constriction injury (CCI) of the sciatic nerve in the rat, in comparison with sham-operated rats. After 3 days from CCI, anandamide or 2-AG levels were significantly enhanced only in the SC or PAG, respectively. After 7 days from CCI, when thermal hyperalgesia and mechanical allodynia are maximal, a strong (1.3-3-fold) increase of both anandamide and 2-AG levels was observed in the PAG, RVM and SC. At this time point, anandamide, but not 2-AG, levels were also enhanced in the DR. PEA levels were significantly decreased in the SC after 3 days, and in the DR and RVM after 7 days from CCI. These data indicate that anandamide and 2-AG, operating at both spinal and supra-spinal levels, are up-regulated during CCI of the sciatic nerve, possibly to inhibit pain. Yet to be developed substances that inhibit both endocannabinoid and PEA inactivation might be useful for the treatment of neuropathic pain.

A mouse model of neuropathic pain consisting of chronic constriction injury (CCI) of the sciatic nerve was used to examine the involvement of reactive oxygen species (ROS) in early spinal cord pro-apoptotic gene over-expression during the development of neuropathic pain. RT-PCR analysis showed increased expression of bax, apoptotic protease-activating factor-1 (apaf-1), and caspase-9 in the dorsal horn spinal cord 3 days after chronic constriction injury of sciatic nerve. Consistent with biomolecular data, a marked increase in TUNEL-positive and caspase-3 active form was observed by 3 days CCI. Administration of phenyl-N-tert-butylnitrone (PBN), a potent ROS scavenger, reduced the development of thermal hyperalgesia and mechanical allodynia at 1 and 3 days post-CCI, and decreased the mRNA levels of bax, apaf-1, and caspase-9. PBN also reduced apoptotic and active Caspase-3 positive profiles in the superficial laminae (I-III) of the spinal cord. This study provides evidence that PBN inhibits over-expression of pro-apoptotic genes and neural apoptosis in the spinal cord dorsal horn induced by early-CCI of the sciatic nerve. These findings suggest that ROS regulate expression of some apoptotic genes which might play a role in the onset of neuropathic pain.

This study investigated the effects of a single administration of 6-(4-methoxyphenyl)-5-methyl-3-pyridinyl-4-isoxazolo[4,5-c]pyridin- 4(5H)-one (MMPIP), a negative allosteric modulator (NAM) of metabotropic glutamate receptor 7 (mGluR7), on pain and on affective and cognitive behavior in neuropathic mice. The activity of pyramidal neurons in the prelimbic cortex (PLC), which respond to stimulation of the basolateral amygdala (BLA) with either excitation or inhibition, was also investigated. The spared nerve injury (SNI) of the sciatic nerve induced, 14 days after surgery, thermal hyperalgesia and mechanical allodynia, reduced open-arm choice in the elevated plus-maze, increased time of immobility in the tail suspension, and increased digging and burying in the marble burying test. Cognitive performance was also significantly compromised in the SNI mice. Spared nerve injury induced phenotypic changes on pyramidal neurons of the PLC; excitatory responses increased, whereas inhibitory responses decreased after BLA stimulation. mGluR7 expression, mainly associated with vesicular glutamate transporter, increased in the hippocampus and decreased in the BLA, PLC, and dorsal raphe in SNI mice. MMPIP increased thermal and mechanical thresholds and open-arm choice. It reduced the immobility in the tail suspension test and the number of marbles buried and of digging events in the marble burying test. MMPIP also improved cognitive performance and restored the balance between excitatory and inhibitory responses of PLC neurons in SNI mice. 7-hydroxy-3-(4-iodophenoxy)-4H-chromen-4-one, XAP044, another selective mGluR7 NAM, reproduced the effects of MMPIP on thermal hyperalgesia, mechanical allodynia, tail suspension, and marble burying test. Altogether, these findings show that mGluR7 NAMs reduce pain responses and affective/cognitive impairments in neuropathic pain conditions. © 2015 International Association for the Study of Pain.

D-Aspartate (D-Asp) is a free D-amino acid found in the mammalian brain with a temporal-dependent concentration based on the postnatal expression of its metabolizing enzyme D-aspartate oxidase (DDO). D-Asp acts as an agonist on NMDA receptors (NMDARs). Accordingly, high levels of D-Asp in knockout mice for Ddo gene (Ddo (-/-)) or in mice treated with D-Asp increase NMDAR-dependent processes. We have here evaluated in Ddo (-/-) mice the effect of high levels of free D-Asp on the long-term plastic changes along the nociceptive pathway occurring in chronic and acute pain condition. We found that Ddo (-/-) mice show an increased evoked activity of the nociceptive specific (NS) neurons of the dorsal horn of the spinal cord (L4-L6) and a significant decrease of mechanical and thermal thresholds, as compared to control mice. Moreover, Ddo gene deletion exacerbated the nocifensive responses in the formalin test and slightly reduced pain thresholds in neuropathic mice up to 7 days after chronic constriction injury. These findings suggest that the NMDAR agonist, D-Asp, may play a role in the regulation of NS neuron electrophysiological activity and behavioral responses in physiological and pathological pain conditions. D-Aspartate (D-Asp) is a free D-amino acid found in the mammalian brain with a temporal-dependent concentration based on the postnatal expression of its metabolizing enzyme D-aspartate oxidase (DDO). D-Asp acts as an agonist on NMDA receptors (NMDARs). Accordingly, high levels of D-Asp in knockout mice for Ddo gene (Ddo -/-) or in mice treated with D-Asp increase NMDAR-dependent processes. We have here evaluated in Ddo -/- mice the effect of high levels of free D-Asp on the long-term plastic changes along the nociceptive pathway occurring in chronic and acute pain condition. We found that Ddo -/- mice show an increased evoked activity of the nociceptive specific (NS) neurons of the dorsal horn of the spinal cord (L4-L6) and a significant decrease of mechanical and thermal thresholds, as compared to control mice. Moreover, Ddo gene deletion exacerbated the nocifensive responses in the formalin test and slightly reduced pain thresholds in neuropathic mice up to 7 days after chronic constriction injury. These findings suggest that the NMDAR agonist, D-Asp, may play a role in the regulation of NS neuron electrophysiological activity and behavioral responses in physiological and pathological pain conditions.

In the ventrolateral periaqueductal gray (PAG), activation of excitatory output neurons projecting monosynaptically to OFF cells in the rostral ventromedial medulla (RVM) causes antinociceptive responses and is under the control of cannabinoid receptor type-1 (CB1) and vanilloid transient receptor potential vanilloid type 1 (TRPV1) receptors. We studied in healthy rats the effect of elevation of PAG endocannabinoid [anandamide and 2-arachidonoylglycerol (2-AG)] levels produced by intra-PAG injections of the inhibitor of fatty acid amide hydrolase URB597 [cyclohexylcarbamic acid-3'-carbamoyl-biphenyl-3-yl ester] on 1) nociception in the "plantar test" and 2) spontaneous and tail-flick-related activities of RVM neurons. Depending on the dose or time elapsed since administration, URB597 (0.5-2.5 nmol/rat) either suppressed or increased thermal nociception via TRPV1 or CB1 receptors, respectively. TRPV1 or cannabinoid receptor agonists capsaicin (6 nmol) and (R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo[1,2,3,-de]-1,4-benzoxazin-6-yl]-1-naphthalenylmethanone mesylate [WIN55,212-2 (4 nmol)] also suppressed or enhanced nociception, respectively. URB597 dose dependently enhanced PAG anandamide and 2-AG levels, with probable subsequent activation of TRPV1/CB1 receptors and only CB1 receptors, respectively. The TRPV1-mediated antinociception and CB1-mediated nociception caused by URB597 correlated with enhanced or reduced activity of RVM OFF cells, suggesting that these effects occur via stimulation or inhibition of excitatory PAG output neurons, respectively. Accordingly, several ventrolateral PAG neurons were found by immunohistochemistry to coexpress TRPV1 and CB1 receptors. Finally, at the highest doses tested, URB597 (4 nmol/rat) and, as previously reported, WIN55,212-2 (25-100 nmol) also caused CB(1)-mediated analgesia, correlating with stimulation (possibly disinhibition) of RVM OFF cells. Thus, endocannabinoids affect the descending pathways of pain control by acting at either CB1 or TRPV1 receptors in healthy rats.

We investigated the role of group III metabotropic glutamate (mGlu) receptors on glutamate and GABA releases at the periaqueductal grey (PAG) level by using in vivo microdialysis in rats. Intra-PAG perfusion of either l -(+)-2-amino-4-phosphonobutyric acid ( l -AP4, 100–300 μM), ( RS )-4-phosphonophenylglycine (( RS )-PPG, 100–300 μM) selective agonists of group III mGlu receptors, or ( S )-3,4-dicarboxyphenylglycine (( S )-3,4-DCPG, 50–100 μM), a selective agonist of mGlu8 receptor, increased glutamate and decreased GABA extracellular concentrations. ( RS )-α-methylserine- O -phosphate (MSOP, 0.5 mM), a selective group III receptor antagonist, perfused in combination with ( S )-3,4-DCPG, l -AP4 or ( RS )-PPG, antagonised the effects induced by these agonists on both extracellular glutamate and GABA values. α-Methyl-3-methyl-4-phosphonophenylglycine (UBP1112, 300 μM), a group III mGlu receptor antagonist, perfused in combination with ( RS )-PPG or ( S )-3,4-DCPG, antagonised the effects induced by these agonists. Intra-PAG perfusion with forskolin (100 μM), an activator of adenylate cyclase, increased dialysate glutamate and GABA levels. Moreover, intra-PAG perfusion with N -[2-( p -bromocinnamyl-amino)ethyl]-5-isoquinolinesulfonamide dihydrochloride (H-89) (100 μM), a protein kinase (PKA) inhibitor, abolished the effect of ( S )-3,4-DCPG on both glutamate and GABA releases. H-89, per se, did not modify glutamate release but reduced extracellular GABA value at the higher dosage used (200 μM). These data suggest that group III mGlu receptors in the PAG modulate the releases of glutamate and GABA conversely. In particular, both the facilitation of glutamate and the inhibition of GABA releases require the participation of coupling to adenylate cyclase and the subsequent activation of the PKA pathway.

Glutamate is the main excitatory neurotransmitter in the central nervous system and as such controls the majority of synapses. Glutamatergic neurotransmission is mediated via ionotropic and metabotropic glutamate receptors (iGluRs and mGluRs). Signaling via mGluRs permits to finely tune, rather than turning on/off, the excitatory neurotransmission as the iGluRs do. Eight mGluRs (mGluR1-8) have been cloned so far, which have been divided into three groups based on sequence homology, pharmacological properties and second messenger signaling. mGluRs are widely expressed both on glia and neurons. On neurons they are located both at postsynaptic (group I) and presynaptic sites (group II and III). Group II and III mGluR stimulation reduces glutamate release, which can prove useful in pathological conditions characterized by elevated glutamatergic neurotransmission which include chronic pain. Indeed, mGluRs are widely distributed on pain neuraxis. The recent development of selective mGluR ligands has permitted investigating the individual role of each mGluR on pain control. The development of (S)-3,4-dicarboxyphenylglycine, a selective mGluR8 agonist, has revealed the mGluR8 role in inhibiting pain and its related affective consequences in chronic pain conditions. mGluR8 proved also to be overexpressed in pain controlling areas during pathological pain guaranteeing the availability of a switch for turning off abnormal pain. Thus, mGluR8 corresponds to an ideal target in designing novel analgesics. This review will focus on the novel insights into the mGluR8 role on pain control, with particular emphasis on the supraspinal descending pathway, an antinociceptive endogenous source, whose activation or disinhibition (via mGluR8) induces analgesia.

The present study investigated the role of metabotropic glutamate receptor subtype 8 (mGluR8) in the dorsal striatum (DS) in modulating thermonociception and rostral ventromedial medulla (RVM) ON and OFF cell activities in conditions of neuropathic pain induced by spared nerve injury (SNI) of the sciatic nerve in rats. The role of DS mGluR8 on mechanical allodynia was also investigated. Intra-DS ( S)-3,4-dicarboxyphenylglycine [( S)-3,4-DCPG], a selective mGluR8 agonist, did not modify the activity of the ON and OFF cells in sham-operated rats. In SNI rats, which showed a reduction of the mechanical withdrawal threshold, intra-DS microinjection of ( S)-3,4-DCPG inhibited the ongoing and tail flick-evoked activity of the ON cells while increasing the activity of the OFF cells. AZ12216052, a selective mGluR8 positive allosteric modulator (PAM), behaved like ( S)-3,4-DCPG in increasing tail flick latency and OFF cell activity and decreasing ON cell activity in SNI rats only but was less potent. VU0155041, a selective mGluR4 PAM, was ineffective in changing thermal nociception and ON and OFF cell activity in both sham-operated and SNI rats. ( S)-3,4-DCPG did not change mechanical withdrawal threshold in sham-operated rats but increased it in SNI rats. Furthermore, a decreased level of mGluR8 gene and immunoreactivity, expressed on GABAergic terminals, associated with a protein increase was found in the DS of SNI rats. These results suggest that stimulation of mGluR8 inhibits thermoceptive responses and mechanical allodynia. These effects were associated with inhibition of ON cells and stimulation of OFF cells within RVM.

The metabotropic glutamate receptor 7 (mGluR7) negative allosteric modulator, 6-(4-methoxyphenyl)-5-methyl-3-pyridin-4-ylisoxazolo[4,5-c]pyridin-4(5H)-one (MMPIP), was locally microinjected into the ventrolateral periaqueductal gray (VL PAG) and the effect on pain responses in formalin and spare nerve injury (SNI) -induced neuropathic pain models was monitored in the rat. The activity of rostral ventromedial medulla (RVM) " pronociceptive" ON and " antinociceptive" OFF cells was also evaluated. Intra-VL PAG MMPIP blocked the first and second phase of nocifensive behaviour in the formalin pain model. MMPIP increased the tail flick latency and simultaneously increased the activity of the OFF cells while inhibiting that of ON cells in rats with SNI of the sciatic nerve. MMPIP failed to modify nociceptive responses and associated RVM ON and OFF cell activity in sham rats. An increase in mGluR7 gene, protein and staining, the latter being associated with vesicular glutamate transporter-positive profiles, has been found in the VL PAG in SNI rats. Blockade of mGluR7 within the VL PAG has an antinociceptive effect in formalin and neuropathic pain models. VL PAG mGluR7 blockade offers a target for dis-inhibiting the VL PAG-RVM pathway and silencing pain in inflammatory and neuropathic pain models. © 2013 Palazzo et al.; licensee BioMed Central Ltd.

Palmitoylethanolamide (PEA) is an endogenous cannabinoid-like compound in the central nervous system, which can modulate several functions in different pathological states, such as inflammation and pain response. We have here investigated the effect of PEA (5-10 mg/kg, intraperitoneally) on mechanical allodynia and thermal hyperalgesia 3 and 7 days following peripheral injection of formalin. Formalin induced a significant decrease of thermal and mechanical threshold in the injected and contralateral paw. PEA chronic treatment (once per day) significantly reduced mechanical allodynia and thermal hyperalgesia in a dose-dependent manner. Consistently, in vivo electrophysiological analysis revealed a significant increase of the duration and frequency, and a rapid decrease in the onset of evoked activity of the spinal nociceptive neurons 7 days after formalin. PEA normalized the electrophysiological parameters in a dose-dependent manner. Moreover, we investigated PEA effect on the glial/microglial phenotypical changes associated with spinal neuronal sensitization. We found that formalin induced a significant microglia and glia activation normalized by PEA, together with increased expression of glial interleukin 10. Finally, primary microglial cell cultures, conditioned with PEA or vehicle, where transplanted in naïve and formalin-treated mice, and nociceptive neurons were recorded. We observed that only PEA-conditioned cells normalized the activity of sensitized nociceptive neurons. In conclusion these data confirm the potent anti-inflammatory and anti-allodynic effect of PEA, and highlight a possible targeted microglial/glial effect of this drug in the spinal cord. © 2013 Bentham Science Publishers.

Background: Salvinorin A (SA), the main active component of Salvia Divinorum, is a non-nitrogenous kappa opioid receptor (KOR) agonist. It has been shown to reduce acute pain and to exert potent antinflammatory effects. This study assesses the effects and the mode of action of SA on formalin-induced persistent pain in mice. Specifically, the SA effects on long-term behavioural dysfuctions and changes in neuronal activity occurring at spinal level, after single peripheral formalin injection, have been investigated. Moreover, the involvement of microglial and glial cells in formalin-induced chronic pain condition and in SA-mediated effects has been evaluated. Results: Formalin induced a significant decrease of mechanical withdrawal threshold at the injected and contralateral paw as well as an increase in the duration and frequency, and a rapid decrease in the onset of evoked activity of the nociceptive neurons 7 days after formalin injection. SA daily treatment significantly reduced mechanical allodynia in KOR and cannabinoid receptor 1 (CB1R) sensitive manner. SA treatment also normalized the spinal evoked activity. SA significantly reduced the formalin-mediated microglia and astrocytes activation and modulated pro and anti-inflammatory mediators in the spinal cord. Conclusion: SA is effective in reducing formalin-induced mechanical allodynia and spinal neuronal hyperactivity. Our findings suggest that SA reduces glial activation and contributes in the establishment of dysfunctions associated with chronic pain with mechanisms involving KOR and CB1R. SA may provide a new lead compound for developing anti-allodynic agents via KOR and CB1R activation.

During neuropathic pain, caspases are activated in the limbic cortex. We investigated the role of TRPV1 channels and glial caspases in the mouse prelimbic and infralimbic (PL-IL) cortex after spared nerve injury (SNI). Reverse transcriptase-polymerase chain reaction, western blots, and immunfluorescence showed overexpression of several caspases in the PL-IL cortex 7 days postinjury. Caspase-3 release and upregulation of AMPA receptors in microglia, caspase-1 and IL-1β release in astrocytes, and upregulation of Il-1 receptor-1, TRPV1, and VGluT1 in glutamatergic neurons, were also observed. Of these alterations, only those in astrocytes persisted in SNI Trpv1(-/-) mice. A pan-caspase inhibitor, injected into the PL-IL cortex, reduced mechanical allodynia, this effect being reduced but not abolished in Trpv1(-/-) mice. Single-unit extracellular recordings in vivo following electrical stimulation of basolateral amygdala or application of pressure on the hind paw, showed increased excitatory pyramidal neuron activity in the SNI PL-IL cortex, which also contained higher levels of the endocannabinoid 2-arachidonoylglycerol. Intra-PL-IL cortex injection of mGluR5 and NMDA receptor antagonists and AMPA exacerbated, whereas TRPV1 and AMPA receptor antagonists and a CB(1) agonist inhibited, allodynia. We suggest that SNI triggers both TRPV1-dependent and independent glutamate- and caspase-mediated cross-talk among IL-PL cortex neurons and glia, which either participates or counteracts pain.

Palmitoylethanolamide (PEA), a peroxisome proliferator-activated receptor-?? (PPAR-??) ligand, exerts antinociceptive and anti-inflammatory effects. PEA (3 and 6 nmol) was microinjected in the ventrolateral periaqueductal grey (VL PAG) of male rats and effects on nociceptive responses and ongoing and tail flick-related activities of rostral ventromedial medulla (RVM) ON and OFF cells were recorded. Intra-PAG microinjection of PEA reduced the ongoing activity of ON and OFF cells and produced an increase in the latency of the nociceptive reaction. These effects were prevented by a selective PPAR-?? antagonist, GW6471 and by a large-conductance Ca(2+)-activated K(+) channel inhibitor, charybdotoxin. Cannabinoid 1 (CB(1)) receptor blockade by AM251 increased the PEA-induced effect both on the ongoing activity of the ON cell and on the latency to tail flick without affecting the effect of PEA on the OFF cell. Conversely, a transient receptor potential vanilloid type 1 (TRPV(1)) blocker, I-RTX, had no effect on the ON cell activity and tail flick latency, whereas it blocked the PEA-induced decrease in ongoing activity of the OFF cell. PEA decreased the burst and increased the latency of tail flick-evoked onset of ON cell activity in a manner antagonised by GW6471 and charybdotoxin. AM251 and I-RTX, instead, enhanced these latter effects. In conclusion, intra-VL PAG PEA induces antinociceptive effects associated with a decrease in RVM ON and OFF cell activities. PPAR-?? receptors mediate, and CB(1) and TRPV(1) receptors antagonise, PEA-induced effects within the PAG-RVM circuitry.

This study has investigated whether the galactosyl ester prodrug of N ω-nitro-L-arginine (NAGAL), shows enhanced analgesic efficacy in healthy mice and in models of visceral and neuropathic pain: the writhing test and the spared nerve injury (SNI), respectively. NAGAL was compared to methyl ester pro-drug of Nω-nitro-l-arginine (L-NAME), a widely exploited non-specific nitric oxide synthase (NOS) inhibitor, for analgesic potential. The writhing test revealed that the ED50 value, along with the 95% confidence limit (CL) was 3.82 (1.77-6.04) mg/kg for NAGAL and, 36.75 (20.07-68.37) mg/kg for L-NAME. Notably, NAGAL elicited a greater anti-allodynic effect than L-NAME did in neuropathic mice. Biomolecular and morphological studies revealed that spared nerve injury increased the expressions of pro-inflammatory enzymes (caspase-1) and two glial cell biomarkers: integrin alpha M (ITGAM) and glial fibrillary acidic protein (GFAP) in the spinal cord. Finally, GLUT-3, an isoform of the hexose transporters capable to bind NAGAL and inducible NOS (iNOS), were found to be over-expressed in the activated astrocytes of the spinal cord of neuropathic mice. NAGAL administration normalized expression levels of these biomarkers. NAGAL showed a greater efficacy in inhibiting visceral pain and allodynia than L-NAME possibly by a greater cell permeation through the hexose transporter which is highly over-expressed by activated glia. © 2011 Elsevier B.V.

Two non-psychoactive cannabinoids, cannabidiol (CBD) and cannabichromene (CBC), are known to modulate in vitro the activity of proteins involved in nociceptive mechanisms, including transient receptor potential (TRP) channels of vanilloid type-1 (TRPV1) and of ankyrin type-1 (TRPA1), the equilibrative nucleoside transporter and proteins facilitating endocannabinoid inactivation. Here we have tested these two cannabinoids on the activity of the descending pathway of antinociception.Electrical activity of ON and OFF neurons of the rostral ventromedial medulla in anaesthetized rats was recorded extracellularly and tail flick latencies to thermal stimuli were measured. CBD or CBC along with various antagonists were injected into the ventrolateral periaqueductal grey.Cannabidiol and CBC dose-dependently reduced the ongoing activity of ON and OFF neurons in anaesthetized rats, whilst inducing antinociceptive responses in the tail flick-test. These effects were maximal with 3 nmol CBD and 6 nmol CBC, and were antagonized by selective antagonists of cannabinoid CB(1) adenosine A(1) and TRPA1, but not of TRPV1, receptors. Both CBC and CBD also significantly elevated endocannabinoid levels in the ventrolateral periaqueductal grey. A specific agonist at TRPA1 channels and a synthetic inhibitor of endocannabinoid cellular reuptake exerted effects similar to those of CBC and CBD.CBD and CBC stimulated descending pathways of antinociception and caused analgesia by interacting with several target proteins involved in nociceptive control. These compounds might represent useful therapeutic agents with multiple mechanisms of action.

Paracetamol is converted to an active metabolite AM404 via fatty acid amide hydrolase (FAAH). The aim of the present study was to ascertain whether a FAAH inhibitor URB597 antagonizes paracetamol analgesic activity (and to asses by this way the role of FAAH in analgesic activity of paracetamol).The interaction between a FAAH inhibitor URB597 and paracetamol was investigated in the writhing test in mice using an isobolographic analysis.URB597 or paracetamol alone and in combinations produced dose-dependent antinociceptive effects. ED50 values were estimated for the individual drugs and an isobologram was constructed. The observed ED50 value for the URB57-paracetamol combination was 0.097 (0.062-0.247) mg/kg. This value did not differ significantly from the theoretical additive ED50 value for the URB597-paracetamol combination which was 0.108 (0.059-0.198) mg/kg. Thus, inhibition of FAAH by URB597 was not followed by the lack of analgesic activity in paracetamol.The present results suggest that the analgesic activity of paracetamol is not dependent solely on FAAH metabolic conversion to AM404 and that paracetamol exerts analgesic activity also by additional mechanisms.

The 6-methoxy-2-phenylimidazo[1,2-b]pyridazine-3-carboxylic acid, DM2, exerts anti-absence activity and blocks Cav3.1 channel, a T-type voltage-dependent Ca2+ channel subtype, in vitro. The current study investigated the effect of intra-ventrolateral periaqueductal grey (VLPAG) administration of DM2 on formalin-induced nocifensive responses in rats. In addition, the effect of intra-VLPAG microinjection of DM2 on the ongoing and tail flick-related activities of rostral ventromedial medulla (RVM) cell population was also investigated. Formalin was injected subcutaneously into the dorsal surface of the hind paws of awake rats. We found that DM2 reduced nocifensive responses in the late phase of the formalin test. Moreover, in the RVM, the intra-VLPAG microinjection of DM2 reduced the ongoing and tail flick-related activity of the nociceptive ON cells, whereas it increased the ongoing activity and reduced the tail flick-induced pause of the antinociceptive OFF cells, consistent with antinociception. Behavioural and electrophysiological effects were reproduced by intra-VLPAG microinjection of ethosuximide, a conventional T-type Ca2+ channel blocker. Finally, DM2 administration did not produce any adverse cardiovascular effects as blood pressure and heart rate remained unchanged. In conclusion, DM2 plays an analgesic role in vivo and changes RVM cell activity, consistent with antinociception. These effects were even more potent than those elicited by ethosuximide treatments.

The transient receptor potential vanilloid-1 (TRPV1) receptor is involved in peripheral and spinal nociceptive processing and is a therapeutic target for pain. We have shown previously that TRPV1 in the ventrolateral periaqueductal gray (VL-PAG) tonically contributes to brain stem descending antinociception by stimulating glutamate release into the rostral ventromedial medulla and off neuron activity. Because both opioid and vanilloid systems integrate and transduce pain sensation in these pathways, we studied the potential interaction between TRPV1 and μ-opioid receptors in the VL-PAG–rostral ventromedial medulla (RVM) system. We found that the TRPV1 agonist, capsaicin, and the μ-receptor agonist [D-Ala(2),N-Me-Phe(4),Gly(5)-ol]enkephalin, when coadministered into the ventrolateral-PAG at doses nonanalgesic per se, produce 1) antinociception in tests of thermal nociception; 2) stimulation of glutamate release into the RVM; and 3) inhibition of on neuron activity in the RVM. These effects were all antagonized by the TRPV1 and opioid receptor antagonists 5′-iodo-resiniferatoxin and naloxone, respectively, thus suggesting the existence of a TRPV1–μ-opioid interaction in the VL-PAG–RVM system. By using double immunofluorescence techniques, we found that TRPV1 and μ-opioid receptors are coexpressed in several neurons of the VL-PAG. These findings suggest that μ-receptor activation not only acts on inhibitory neurons to disinhibit PAG output neurons but also interacts with TRPV1 activation at increasing glutamate release into the RVM, possibly by acting directly on PAG output neurons projecting to the RVM.

We have studied the involvement of the N-methyl-D-aspartate receptor (NMDAR) glycine site and the strychnine-sensitive glycine receptor (GlyR) in the ventrolateral periaqueductal gray (VL-PAG) on nociceptive behavior (tail flick) and pain-related changes on neuronal activity in the rostral ventromedial medulla (RVM). Glycine or D-serine increased the tail-flick latency, reduced OFF-cell pause, and delayed its onset and increased the time between the onset of the OFF-cell pause and the tail withdrawal. Conversely, they decreased the ongoing activity of the ON cell, the tail-flick-induced ON-cell firing, whereas they delayed the onset of increased tail-flick-induced ON-cell firing. Also, glycine or D-serine reduced the interval between the onset of the increased ON-cell firing and tail withdrawal. Whereas 7-Cl-kynurenic acid (7-Cl-KYN) prevented such effects, strychnine did not do so. A higher dose of 7-Cl-KYN or strychnine was per se able to reduce or increase tail-flick latency and increase or reduce ON-cell activities, respectively. A higher dose of glycine was hyperalgesic in the presence of 7-Cl-KYN, whereas such an effect was prevented by strychnine. These data suggest 1) a dual role of glycine in producing hyperalgesia or analgesia by stimulating the GlyR or the NMDARs within the VL-PAG, respectively; 2) consistently that RVM ON and OFF cells display opposite firing patterns to the stimulation of the VL-PAG NMDAR glycine site and GlyR activation; and 3) a tonic role of these receptors within the VL-PAG-RVM antinociceptive descending pathway., This work was supported by Ministero dell'Università e della Ricerca (Italy) Grant PRIN 2005 and Ministry of Education and Science (Spain) Grant SAF2005-3185.

The effect of the non-selective, 1-aminoindan-1,5-dicarboxylic acid (AIDA), and selective (3,4-dihydro-2H-pyrano[2,3-b]quinolin-7-yl)-(cis-4-methoxycyclohexyl) methanone (JNJ16259685), metabotropic glutamate subtype 1 (mGlu1) receptor antagonists, on rat sciatic nerve chronic constrictive injury (CCI)-induced hyperalgesia, allodynia, spinal dorsal horn apoptosis, and gliosis was examined at 3 and 7 days post-injury. RT-PCR analysis showed increased expression of bax, apoptotic protease-activating factor-1 (apaf-1), nestin, GFAP, and caspase-7 mRNA in the dorsal horn spinal cord by 3 days post-CCI. At 7 days post-CCI, only over-expression of bcl-2, nestin and GFAP mRNA was observed. Administration of AIDA reduced thermal hyperalgesia and mechanical allodynia at 3 and 7 days post-CCI; administration of JNJ16259685 reduced thermal hyperalgesia at 3 and 7 days post-CCI, but not mechanical allodynia. AIDA decreased the mRNA levels of bax, apaf-1, GFAP and caspase-7 genes. JNJ16259685 increased the mRNA levels of bcl-2 and GFAP gene, and decreased APAF-1 and caspases-7 genes. Inhibiting mGlu1 receptors also reduced TUNEL-positive profiles and immunohistochemical reactivity for caspase-7. We report here that despite inhibiting CCI-induced over-expression of pro-apoptotic genes in the spinal cord dorsal horn, the selective mGlu1 receptor antagonist JNJ16259685 exerted only a slight and transient allodynic effect. Moreover, JNJ16259685, but not the non-selective AIDA, increased astrogliosis which may account for its decreased analgesic efficacy. This study provides evidence that the contemporary and partial blockade of group I and likely ionotropic glutamate receptors may be a more suitable therapy than selective blockade of mGlu1 subtype receptors condition to decrease neuropathic pain symptoms. © 2008 Elsevier Ltd. All rights reserved.

Activation of transient receptor potential vanilloid-1 (TRPV1) channels in the periaqueductal gray (PAG) activates OFF antinociceptive neurons of the rostral ventromedial medulla (RVM). We examined in rats the effect of intra-ventrolateral (VL)-PAG injections of TRPV1 agonists and antagonists on the nocifensive response to heat in the plantar test, neurotransmitter (glutamate and GABA) release in the RVM, and spontaneous and tail flick-related activities of RVM neurons. The localization of TRPV1 in VL-PAG and RVM neurons was examined using various markers of glutamatergic and GABAergic neurons. Intra-VL-PAG injection of capsaicin increased the threshold of thermal pain sensitivity, whereas the selective TRPV1 antagonist 5′-iodo-resiniferatoxin (I-RTX) facilitated nociceptive responses, and blocked capsaicin analgesic effect at a dose inactive per se. Intra-VL PAG capsaicin evoked a robust release of glutamate in RVM microdialysates. I-RTX, at a dose inactive per se, blocked the effect of capsaicin, and inhibited glutamate release at a higher dose. Antinociception and hyperalgesia induced by capsaicin and I-RTX, respectively, correlated with enhanced or reduced activity of RVM OFF cells. Immunohistochemical analyses suggested that several TRPV1-immunoreactive (ir) neurons in both the VL-PAG and RVM are glutamatergic and surrounded by glutamatergic and GABAergic terminals. Our data suggest that VL-PAG neurons respond to TRPV1 stimulation by releasing glutamate into the RVM, thereby activating OFF cells and producing analgesia. The results obtained with the TRPV1 antagonist alone suggest that this pathway is tonically activated by endovanilloids.

We used a model of neuropathic pain consisting of rats with chronic constriction injury (CCI) of the sciatic nerve, in order to investigate whether endocannabinoid levels are altered in the dorsal raphe (DR) and to assess the effect of repeated treatment with (R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl]-1-naphthalenylmethanone mesylate, a synthetic cannabinoid agonist, or N-(4-hydroxyphenyl)-5Z,8Z,11Z,14Z-eicosatetraenamide (AM404), an inhibitor of endocannabinoid reuptake, on DR serotonergic neuronal activity and on behavioural hyperalgesia. CCI resulted in significantly elevated anandamide but not 2-arachidonoylglycerol levels in the DR. Furthermore, as well as thermal and mechanical hyperalgesia, CCI caused serotonergic hyperactivity (as shown by the increase of basal activity of serotonergic neurones, extracellular serotonin levels and expression of 5-HT1A receptor gene). Repeated treatment with either (R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo[ 1,2,3-de]-1,4-benzoxazin-6-yl]-1-naphthalenylmethanone mesylate or AM404 reverted the hyperalgesia and enhanced serotonergic activity induced by CCI in a way attenuated by N-piperidino-5-(4-chlorophenyl)-1-(2,4dichlorophenyl)-4-methyl-3- pyrazolecarboxamide, a selective cannabinoid subtype 1 (CB1) receptor antagonist. Despite the elevated levels of anandamide following CCI, N-piperidino-5-(4-chlorophenyl)-1-(2,4dichlorophenyl)-4-methyl-3-pyrazolecarboxamide did not produce hyperalgesia or any other effect on serotonergic neuronal activity when administered alone. Furthermore, the effects of AM404 were not accompanied by an increase in endocannabinoid levels in the DR. In conclusion, following CCI of the sciatic nerve, the endocannabinoid and serotonergic systems are activated in the DR, where repeated stimulation of CB1 receptors with exogenous compounds restores DR serotonergic activity, as well as thermal and mechanical nociceptive thresholds, to pre-surgery levels. However, an elevated level of endogenous anandamide in the DR does not necessarily contribute to the CB1-mediated tonic control of analgesia and serotonergic neuronal activity.

In this study we have investigated the role of periaqueductal grey prostaglandin receptors in formalin-induced hyperalgesia in mice. Glutamate and GABA release changes have been monitored by in vivo microdialysis. Intra-periaqueductal grey microinjections of misoprostol, a non-selective prostaglandin receptor agonist, increased nociceptive responses in the formalin test only during the late phase. Prostanoid EP(1) (L-335677), EP(2) (AH 6809), EP(3) (L-826266) and EP(4) (L-161982) receptor antagonists prevented the nociceptive response induced by misoprostol in formalin-injected mice. Prostanoid EP(1), EP(2), EP(3) and EP(4) antagonists reduced, per se, the late hyperalgesic phase. Intra-periaqueductal grey perfusion with misoprostol increased periaqueductal grey glutamate, whereas it produced an increase followed by a decrease in GABA. Likewise, formalin increased glutamate and produced a biphasic response on GABA. When misoprostol was perfused in combination with the peripheral injection of formalin, we observed an increase of glutamate and an increase followed by a stronger decrease in GABA release. These data show that periaqueductal grey prostaglandin receptor stimulation increased formalin-induced nociceptive response in the late phase by increasing glutamate release and by producing a biphasic change in GABA release.

In this study, we evaluated the effects of 2-methyl-6-(phenylethynyl)-pyridine (MPEP), a selective antagonist of metabotropic glutamate subtype 5 receptors (mGlu(5)), delivered through different paths on dorsal raphe serotonin (5-HT) and on thermoceptive responses in rats with inflammatory pain. Intraplantar formalin and carrageenan increased 5-HT (137+/-11% and 212+/-6% of pre-injection baseline, respectively) and reduced nociceptive threshold (23+/-7% and 19+/-3% of pre-injection baseline, respectively). MPEP (2 mg/kg i.p.) further enhanced formalin and carrageenan-induced 5-HT increases (180+/-11% and 260+/-12% of pre-injection baseline, respectively) and reduced thermal hyperalgesia (71+/-8% and 80+/-10% of pre-injection baseline, respectively). MPEP (1 mM) through microdialytic probe into the dorsal raphe did not change formalin- or carrageenan-induced 5-HT increases (147+/-10% and 189+/-10% of pre-injection baseline, respectively) and thermal hyperalgesia (35+/-8% and 25+/-9% of pre-injection baseline, respectively). Finally, MPEP (30 nmol/rat) into the hind paw reduced the formalin- and carrageenan-induced 5-HT increase (108+/-3% and 126+/-7% of pre-injection baseline, respectively) and thermal hyperalgesia (77+/-6% and 117+/-7% of pre-injection baseline, respectively). Dorsal raphe serotonergic neurons activity increased following a peripherally induced inflammatory injury. In these conditions, peripheral but not dorsal raphe mGlu(5) receptors blockade prevented over activation of dorsal raphe serotonergic neurons and reversed thermal hyperalgesia.

We used rats with a sciatic nerve chronic constrictive injury (CCI) and combined behavioural, molecular and morphological approaches to assess the involvement of mGlu5 receptors in neuropathic pain-associated hyperalgesia and spinal cord neuron apoptosis. Mechanical and thermal hyperalgesia developed 2–3 days after surgery. Morphological changes in the ipsilateral L4–L5 lamina II consisted of: (i) cell loss (38 +/- 5%), (ii) increased TUNEL-positive profiles, (iii) decreased SP-immunoreactive primary afferents, and (iv) reactive gliosis. Molecular expression data suggested a bi-phasic response of bcl-2 family genes in CCI. An early (2–3 days post-CCI) E2F1- and p53-independent apoptosis appeared in the spinal cord as the pro-apoptotic bax gene increased (320 +/- 19%), followed by an increased expression of the anti-apoptotic bcl-2 and bcl-xL genes (60 +/- 11% and 110 +/- 15%, respectively) 7 days from CCI. The selective mGlu5 receptor antagonist, MPEP (2 mg/kg i.p. twice daily), prevented the development of thermal hyperalgesia and transiently reduced mechanical hyperalgesia. Despite the MPEP treatment, which normalised bax=bcl-2 and bcl-xL=bcl-xS ratios at all times post-CCI, mechanical hyperalgesia reappeared by 7 days after CCI. Similarly, MPEP was cytoprotective at 3, but not 7 days post-CCI. This study shows that: (a) spinal cord neuron loss may be triggered by a p53- and E2F1-independent apoptosis in lamina II with the participation of glutamate mGlu5 receptors, (b) these receptors seem to be involved transiently, as their blockade was no longer protective by 7 days CCI, and (c) this delayed cell death occurred in the absence of Bax activation, suggesting the involvement of an alternative death pathway.

In this study, we investigated the effects of group I metabotropic glutamate (mglu) receptor ligands on glutamate and gamma-aminobutyric acid (GABA) extracellular concentrations at the periaqueductal grey level by using in vivo microdialysis. An agonist of group I mglu receptors, (S)-3,5-dihydroxyphenylglycine [(S)-3,5-DHPG, 1 and 2 mM], as well as a selective agonist of mglu(5) receptors, (RS)-2-chloro-5-hydroxyphenylglycine (CHPG, 2 and 4 mM), both increased dialysate glutamate and GABA concentrations. 7-(Hydroxyimino)cyclopropa-[b]-chromen-1alpha-carboxylate ethyl ester (CPCCOEt, 1 mM), a selective mglu(1) receptor antagonist, and 2-methyl-6-(phenylethynyl)pyridine (MPEP, 0.5 mM), a selective mglu(5) receptor antagonist, perfused in combination with DHPG, antagonized the effect induced by DHPG on the extracellular glutamate and GABA concentrations. MPEP (0.5 mM), perfused in combination with CHPG, antagonized the increased glutamate and GABA extracellular levels induced by CHPG. MPEP (1 mM) decreased the extracellular concentrations of glutamate but did not modify the dialysate GABA concentrations. Moreover, as the intra-periaqueductal grey perfusion of (RS)-3-(2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid [(RS)-CPP, 100 microM], a selective N-methyl-D-aspartate (NMDA) glutamate receptor antagonist, did not change the extracellular concentrations of glutamate, this suggests that the MPEP-induced decrease in glutamate is not a consequence of NMDA receptor blockade. These data show that group I mglu receptors in the periaqueductal grey may modulate the release of glutamate and GABA in awake, freely moving rats. In particular, mglu(5), but not mglu(1), receptors seem to be functionally active on glutamate terminals.

Adenosine reduces opioid withdrawal symptoms by activating A(1) adenosine receptors, probably by inhibiting excitatory amino acid release. Since blockade of A(2A) adenosine receptors seems to enhance dopaminergic striatopallidal transmission, we evaluated the role of the purinergic system in the opiate withdrawal syndrome by using two A(1) receptor agonists [ N(6)-cyclohexyladenosine, CHA and 2-chloro- N(6)-cyclopentyladenosine, CCPA], and two A(2A) receptor antagonists (SCH 58261 and 8-(3-chlorostyryl)caffeine, CSC). Male adult rats received increasing doses of morphine sulphate suspended in 5 ml/kg of a sustained release preparation (40-100 mg/kg s.c.) daily for 4 days and 20 h after the last administration, the withdrawal syndrome was evoked by naloxone (5 mg/kg i.p.). Animals were observed for 30 min for signs of opiate withdrawal. Other groups of rats were implanted with concentric probes for microdialysis and dopamine levels were measured in the nucleus accumbens. CHA and CCPA (0.05, 0.1 or 0.5 mg/kg i.p.) significantly reduced "wet-dog" shakes, diarrhoea, teeth chattering, jumping and writhing. SCH 58261 and CSC (0.1, 0.5 or 1 mg/kg i.p.), given 10 min before naloxone, also reduced signs of opiate withdrawal. CHA plus SCH 58261 and CCPA plus CSC greatly enhanced the reduction of withdrawal signs observed with CHA and CCPA or CSC and SCH 58261 alone. In vivo microdialysis showed that naloxone significantly decreased DA release; this effect was prevented by pretreatment with systemic SCH 58261 and CSC, but not with CHA and CCPA. Our results demonstrate that A(1) and A(2A) adenosine receptors mediate the effect induced by adenosine in opiate withdrawal syndrome and suggest that adenosine A(1) agonists and adenosine A(2A) antagonists may be beneficial in the treatment of this syndrome.

This study investigates the effect of microinjections of capsaicin in the periaqueductal grey matter of rats on nociceptive behaviour and the possible interactions with NMDA and mGlu receptors. Intra-periaqueductal grey microinjection of capsaicin (1-3-6 nmol/rat) increased the latency of the nociceptive reaction in the plantar test. This effect was prevented by pretreatment with capsazepine (6 nmol/rat), which had no effect per se on the latency of the nociceptive reaction. 7-(Hydroxyimino)cyclopropa[b]chromen-1alpha-carboxylate ethyl ester (CPCCOEt, 50 nmol/rat) and 2-Methyl-6-(phenylethynyl)pyridine (MPEP, 50 nmol/rat), antagonists of mGlu(1) and mGlu(5) receptors, respectively, completely blocked the effect of capsaicin. Similarly, pretreatment with DL-2-Amino-5-phosphonovaleric acid (DL-AP5, 5 nmol/rat) and riluzole (4 nmol/rat), an NMDA receptor antagonist and a voltage-dependent Na(+) channels blocker which inhibits glutamate release, respectively, completely antagonized the effect of capsaicin. However, pretreatment with (2S)-alpha-Ethylglutamic acid (30 nmol/rat) and (RS)-alpha-Methylserine-O-phosphate (MSOP, 30 nmol/rat), antagonists of group II and group III mGlu receptors, respectively, had no effects on capsaicin-induced analgesia. Similarly, pretreatment with N-(piperidin-1-yl)-5-(4-chlophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (SR 141716A, 5 pmol/rat), a selective cannabinoid CB(1) receptor antagonist, did not affect the capsaicin-induced antinociception. In conclusion, this study shows that capsaicin might produce antinociception at the periaqueductal grey level by increasing glutamate release, which activates postsynaptic group I mGlu and NMDA receptors.

The aim of the study was to examine whether endothelin-1 (ET-1) injected into dorsolateral periaqueductal gray (PAG) area of mice produces antinociception. ET-1, from 1 to 4 pmol/mouse, induced antinociceptive effect in a dose-dependent manner. This antinociceptive effect was prevented by NMDA receptor antagonists (2-APV and MK-801) injected in the same area (2-APV) or by intraperitoneal route (MK-801). CNQX, a non-NMDA receptor antagonist, did not inhibit the ET-1 effects. Prazosin, an α 1 -adrenergic blocking agent, also prevented the ET-1 antinociceptive effect. We suggest that the activation of NMDA glutamatergic receptors in the PAG area may be a necessary step for ET-1 induced antinociception.

Background: Neuropathic pain is a chronic disease resulting from dysfunction within the “pain matrix”. The basolateral amygdala (BLA) can modulate cortical functions and interactions between this structure and the medial prefrontal cortex (mPFC) are important for integrating emotionally salient information. In this study, we have investigated the involvement of the transient receptor potential vanilloid type 1 (TRPV1) and the catabolic enzyme fatty acid amide hydrolase (FAAH) in the morphofunctional changes occurring in the pre-limbic/infra-limbic (PL/IL) cortex in neuropathic rats. Results: The effect of N-arachidonoyl-serotonin (AA-5-HT), a hybrid FAAH inhibitor and TPRV1 channel antagonist, was tested on nociceptive behaviour associated with neuropathic pain as well as on some phenotypic changes occurring on PL/IL cortex pyramidal neurons. Those neurons were identified as belonging to the BLA-mPFC pathway by electrical stimulation of the BLA followed by hind-paw pressoceptive stimulus application. Changes in their spontaneous and evoked activity were studied in sham or spared nerve injury (SNI) rats before or after repeated treatment with AA-5-HT. Consistently with the SNI-induced changes in PL/IL cortex neurons which underwent profound phenotypic reorganization, suggesting a profound imbalance between excitatory and inhibitory responses in the mPFC neurons, we found an increase in extracellular glutamate levels, as well as the upregulation of FAAH and TRPV1 in the PL/IL cortex of SNI rats. Daily treatment with AA-5-HT restored cortical neuronal activity, normalizing the electrophysiological changes associated with the peripheral injury of the sciatic nerve. Finally, a single acute intra-PL/IL cortex microinjection of AA-5-HT transiently decreased allodynia more effectively than URB597 or I-RTX, a selective FAAH inhibitor or a TRPV1 blocker, respectively. Conclusion: These data suggest a possible involvement of endovanilloids in the cortical plastic changes associated with peripheral nerve injury and indicate that therapies able to normalize endovanilloid transmission may prove useful in ameliorating the symptoms and central sequelae associated with neuropathic pain.