Blockade of neurotransmitter release by botulinum neurotoxin type A (BoNT A ) underlies the severe neuroparalytic symptoms of human botulism, which can last a few years. The structural basis for this remarkable persistence remains unclear. Herein, recombinant BoNT A was found to match the neurotoxicity of that from Clostridium botulinum, producing persistent cleavage of synaptosomal-associated protein of 25 kDa (SNAP-25) and neuromuscular paralysis. When two leucines near the C terminus of the protease light chain of A (LC A ) were mutated, its inhibition of exocytosis was followed by fast recovery of intact SNAP-25 in cerebellar neurons and neuromuscular transmission in vivo. Deletion of 6 -7 N terminus residues diminished BoNT A activity but did not alter the longevity of its SNAP-25 cleavage and neuromuscular paralysis. Furthermore, genetically fusing LC E to a BoNT A enzymically inactive mutant (BoTIM A ) yielded a novel LC E -BoTIM A protein that targets neurons, and the BoTIM A moiety also delivers and stabilizes the inhibitory LC E , giving a potent and persistent cleavage of SNAP-25 with associated neuromuscular paralysis. Moreover, its neurotropism was extended to sensory neurons normally insensitive to BoNT E . LC E-BoTIM A (AA) with the above-identified dileucine mutated gave transient neuromuscular paralysis similar to BoNT E , reaffirming that these residues are critical for the persistent action of LC E -BoTIM A as well as BoNT A . LC EBoTIM A inhibited release of calcitonin gene-related peptide from sensory neurons mediated by transient receptor potential vanilloid type 1 and attenuated capsaicin-evoked nociceptive behavior in rats, following intraplantar injection. Thus, a long acting, versatile composite toxin has been developed with therapeutic potential for pain and conditions caused by overactive cholinergic nerves. Botulinum neurotoxins (BoNTs)3 inhibit transmitter release from peripheral cholinergic neurons causing the lifethreatening flaccid paralysis underlying botulism (1). The most potent biological substances, their estimated lethal doses (LD 50 ) in humans are between 0.1 and 1 ng/kg. Seven BoNT serotypes (A-G), produced by Clostridium botulinum, are synthesized as pro-form single chain proteins (SC, M r ϳ150,000) and converted by either Clostridial or tissue proteases into fully active dichain (DC) forms, consisting of a protease domain (LC; M r ϳ50,000) linked to a heavy chain (HC; M r ϳ100,000) through disulfide and noncovalent bonds. BoNT A preferentially enters cholinergic nerve endings by binding via the C-terminal half of their HC to a membrane acceptor, a lumenal domain of synaptic vesicle protein 2 (2, 3). On the other hand, type E only binds the glycosylated synaptic vesicle protein 2 A/B isoforms (4), which are sparsely expressed on sensory neurons, explaining its lack of effects on trigeminal ganglionic neurons (TGNs) (5). These toxins undergo acceptor-mediated endocytosis (6, 7) with translocation of the LCs into the cytosol through a channel formed by the N-terminal half...

The intensity and severity of perceived pain does not correlate consistently with the degree of peripheral or central nervous system tissue damage or with the intensity of primary afferent or spinal nociceptive neurone activity. In this respect, the modulation of pain by emotion and context is now widely recognized. In particular, stress, fear and anxiety exert potent, but complex, modulatory influences on pain. Stress can either suppress pain (stress-induced analgesia) or exacerbate it (stress-induced hyperalgesia; SIH) depending on the nature, duration and intensity of the stressor. Herein, we review the methods and models used to study the phenomenon of SIH in rodents and humans and then present a detailed discussion of our current understanding of neural substrates and neurobiological mechanisms. The review provides perspectives and challenges for the current and future treatment of pain and the co-morbidity of pain with stress-related psychiatric disorders including anxiety and depression.

BACKGROUND AND PURPOSEEndocannabinoids in the midbrain periaqueductal grey (PAG) modulate nociception and unconditioned stress-induced analgesia; however, their role in fear-conditioned analgesia (FCA) has not been examined. The present study examined the role of the endocannabinoid system in the dorsolateral (dl) PAG in formalin-evoked nociceptive behaviour, conditioned fear and FCA in rats. EXPERIMENTAL APPROACHRats received intra-dlPAG administration of the CB1 receptor antagonist/inverse agonist rimonabant, or vehicle, before re-exposure to a context paired 24 h previously with foot shock. Formalin-evoked nociceptive behaviour and fear-related behaviours (freezing and 22 kHz ultrasonic vocalization) were assessed. In a separate cohort, levels of endocannabinoids [2-arachidonoyl glycerol (2-AG) and N-arachidonoyl ethanolamide (anandamide; AEA)] and the related N-acylethanolamines (NAEs) [N-palmitoyl ethanolamide (PEA) and N-oleoyl ethanolamide (OEA)] were measured in dlPAG tissue following re-exposure to conditioned context in the presence or absence of formalin-evoked nociceptive tone. KEY RESULTSRe-exposure of rats to the context previously associated with foot shock resulted in FCA. Intra-dlPAG administration of rimonabant significantly attenuated FCA and fear-related behaviours expressed in the presence of nociceptive tone. Conditioned fear without formalin-evoked nociceptive tone was associated with increased levels of 2-AG, AEA, PEA and OEA in the dlPAG. FCA was specifically associated with an increase in AEA levels in the dlPAG. CONCLUSIONS AND IMPLICATIONSConditioned fear to context mobilises endocannabinoids and NAEs in the dlPAG. These data support a role for endocannabinoids in the dlPAG in mediating the potent suppression of pain responding which occurs during exposure to conditioned aversive contexts. BJPBritish Journal of Pharmacology DOI:10.1111DOI:10. /j.1476DOI:10. -5381.2011 British Journal of Pharmacology (2012) IntroductionThough pain is part of a global defence response initiated upon exposure to noxious stimuli, maladaptive persistent/ chronic pain is a major unmet clinical need. Conditioned fear is known to suppress nociceptive behaviour potently, resulting in fear-conditioned analgesia (FCA), the phenomenon by which re-exposure of an animal to a context previously paired with an aversive stimulus (e.g. foot shock) results in conditional analgesia (Ford and Finn, 2008;Butler and Finn, 2009). A large body of evidence suggests overlap in the neural substrates mediating pain and conditioned fear. Recent studies have also described significant co-morbidity of anxiety disorders with persistent pain conditions (Asmundson and Katz, 2009) and altered pain processing in patients with anxiety disorders, including post-traumatic stress disorder (Geuze et al., 2007;Kraus et al., 2009). In light of this evidence, detailed understanding of the neurobiology underpinning the relationship between fear and pain is of fundamental physiological and potential therapeutic significance. The periaqueductal...

Pain is both a sensory and an emotional experience, and is subject to modulation by a number of factors including genetic background modulating stress/affect. The Wistar-Kyoto (WKY) rat exhibits a stress-hyper-responsive and depressive-like phenotype and increased sensitivity to noxious stimuli, compared with other rat strains. Here, we show that this genotype-dependent hyperalgesia is associated with impaired pain-related mobilisation of endocannabinoids and transcription of their synthesising enzymes in the rostral ventromedial medulla (RVM). Pharmacological blockade of the Cannabinoid1 (CB1) receptor potentiates the hyperalgesia in WKY rats, whereas inhibition of the endocannabinoid catabolising enzyme, fatty acid amide hydrolase, attenuates the hyperalgesia. The latter effect is mediated by CB1 receptors in the RVM. Together, these behavioural, neurochemical, and molecular data indicate that impaired endocannabinoid signalling in the RVM underpins hyper-responsivity to noxious stimuli in a genetic background prone to heightened stress/affect.

BackgroundSeveral factors contribute to the deterioration in synaptic plasticity which accompanies age and one of these is neuroinflammation. This is characterized by increased microglial activation associated with increased production of proinflammatory cytokines like interleukin-1β (IL-1β). In aged rats these neuroinflammatory changes are associated with a decreased ability of animals to sustain long-term potentiation (LTP) in the dentate gyrus. Importantly, treatment of aged rats with agents which possess anti-inflammatory properties to decrease microglial activation, improves LTP. It is known that endocannabinoids, such as anandamide (AEA), have anti-inflammatory properties and therefore have the potential to decrease the age-related microglial activation. However, endocannabinoids are extremely labile and are hydrolyzed quickly after production. Here we investigated the possibility that inhibiting the degradation of endocannabinoids with the fatty acid amide hydrolase (FAAH) inhibitor, URB597, could ameliorate age-related increases in microglial activation and the associated decrease in LTP.MethodsYoung and aged rats received subcutaneous injections of the FAAH inhibitor URB597 every second day and controls which received subcutaneous injections of 30% DMSO-saline every second day for 28 days. Long-term potentiation was recorded on day 28 and the animals were sacrificed. Brain tissue was analyzed for markers of microglial activation by PCR and for levels of endocannabinoids by liquid chromatography coupled to tandem mass spectrometry.ResultsThe data indicate that expression of markers of microglial activation, MHCII, and CD68 mRNA, were increased in the hippocampus of aged, compared with young, rats and that these changes were associated with increased expression of the proinflammatory cytokines interleukin (IL)-1β and tumor necrosis factor-α (TNFα) which were attenuated by treatment with URB597. Coupled with these changes, we observed an age-related decrease in LTP in the dentate gyrus which was partially restored in URB597-treated aged rats. The data suggest that enhancement of levels of endocannabinoids in the brain by URB597 has beneficial effects on synaptic function, perhaps by modulating microglial activation.