Weredeselam M. Olango

A dileucine in the protease of botulinum toxin A underlies its long-lived neuroparalysis: transfer of longevity to a novel potential therapeutic.

Blockade of neurotransmitter release by botulinum neurotoxin type A (BoNTA) 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 BoNTA 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 (LCA) 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 BoNTA activity but did not alter the longevity of its SNAP-25 cleavage and neuromuscular paralysis. Furthermore, genetically fusing LCE to a BoNTA enzymically inactive mutant (BoTIMA) yielded a novel LCE-BoTIMA protein that targets neurons, and the BoTIMA moiety also delivers and stabilizes the inhibitory LCE, giving a potent and persistent cleavage of SNAP-25 with associated neuromuscular paralysis. Moreover, its neurotropism was extended to sensory neurons normally insensitive to BoNTE. LCE-BoTIMA(AA) with the above-identified dileucine mutated gave transient neuromuscular paralysis similar to BoNTE, reaffirming that these residues are critical for the persistent action of LCE-BoTIMA as well as BoNTA. LCE-BoTIMA 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.

The endocannabinoid system in the rat dorsolateral periaqueductal grey mediates fear-conditioned analgesia and controls fear expression in the presence of nociceptive tone

BACKGROUND AND PURPOSE Endocannabinoids 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.

The fatty acid amide hydrolase inhibitor URB597 exerts anti-inflammatory effects in hippocampus of aged rats and restores an age-related deficit in long-term potentiation

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.

Impaired endocannabinoid signalling in the rostral ventromedial medulla underpins genotype-dependent hyper-responsivity to noxious stimuli.

Summary Impaired endocannabinoid signalling in the rostral ventromedial medulla underpins hyper‐responsivity to a noxious inflammatory stimulus in the Wistar–Kyoto rat, a genetic background prone to heightened stress/affect. ABSTRACT 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.

Neurobiology of Stress-Induced Hyperalgesia

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.

Evidence for a role of GABAergic and glutamatergic signalling in the basolateral amygdala in endocannabinoid-mediated fear-conditioned analgesia in rats

Summary CB1 receptors in the basolateral amygdala facilitate the expression of fear‐conditioned analgesia through a mechanism involving the modulation of GABAergic and glutamatergic signalling. Abstract The basolateral amygdala (BLA) is a key substrate facilitating the expression of fear‐conditioned analgesia (FCA). However, the neurochemical mechanisms in the BLA which mediate this potent suppression of pain responding during fear remain unknown. The present study investigated the role of cannabinoid1 (CB1) receptors and interactions with GABAergic (GABAA receptor) and glutamatergic (metabotropic glutamate receptor type 5; mGluR5) signalling in the BLA in formalin‐evoked nociceptive behaviour and FCA in rats. Reexposure to a context previously paired with foot shock significantly reduced formalin‐evoked nociceptive behaviour. Systemic or intra‐BLA microinjection of the CB1 receptor antagonist/inverse agonist AM251 prevented this expression of FCA, while injection of AM251 into the central nucleus of the amygdala did not. The suppression of FCA by systemic AM251 administration was partially attenuated by intra‐BLA administration of either the GABAA receptor antagonist bicuculline or the mGluR5 antagonist 2‐methyl‐6‐(phenylethynyl) pyridine, (MPEP). Bilateral microinjection of MPEP, but not bicuculline, alone into the BLA enhanced formalin‐evoked nociceptive behaviour. Postmortem analyses revealed that FCA was associated with a significant increase in tissue levels of anandamide in the BLA side contralateral to intraplantar formalin injection. In addition, fear‐conditioned rats exhibited a robust formalin‐induced increase in levels of 2‐arachidonyl glycerol and N‐palmitoylethanolamide in the ipsilateral and contralateral BLA, respectively. These data suggest that CB1 receptors in the BLA facilitate the expression of FCA, through a mechanism which is likely to involve the modulation of GABAergic and glutamatergic signalling.

Effects of intra-basolateral amygdala administration of rimonabant on nociceptive behaviour and neuronal activity in the presence or absence of contextual fear

The basolateral amygdala (BLA) contains a high density of cannabinoid CB1 receptors and is critically involved in pain and fear‐related behaviour. We investigated the effects of bilateral intra‐BLA administration of the CB1 receptor antagonist/inverse agonist, rimonabant, on formalin‐evoked nociceptive behaviour, fear‐conditioned behaviour including analgesia, and associated brain regional alterations in Fos expression in rats. Intra‐BLA administration of rimonabant significantly reduced formalin‐evoked nociceptive behaviour in the absence, but not presence, of conditioned fear. Rimonabant attenuated a formalin‐evoked reduction in freezing while emitting 22 kHz ultrasonic vocalisation in the early part of the fear expression trial. Formalin‐evoked nociceptive behaviour was associated with increased Fos immunoreactivity (FI) in the CA2/3 region of the hippocampus and rostral ventromedial medulla, effects attenuated by intra‐BLA rimonabant. Formalin also decreased FI in the cingulate cortex, an effect which was not observed in fear‐conditioned rats. Contextually‐induced fear was associated with increased FI in the dorsal caudal periaqueductal grey in the absence, but not presence, of formalin‐evoked nociceptive tone. In conclusion, bilateral intra‐BLA administration of rimonabant reduces nociceptive behaviour in a model of tonic, persistent inflammatory pain, an effect associated with reduced activation of neurons in the CA2/3 hippocampus and rostral ventromedial medulla. The data also provide evidence for differential pain‐ and fear‐related brain regional activity in the presence or absence of contextually‐induced aversion and nociceptive tone.

A role for PPARα in the medial prefrontal cortex in formalin‐evoked nociceptive responding in rats

The nuclear hormone receptor, PPARα, and its endogenous ligands, are involved in pain modulation. PPARα is expressed in the medial prefrontal cortex (mPFC), a key brain region involved in both the cognitive‐affective component of pain and in descending modulation of pain. However, the role of PPARα in the mPFC in pain responding has not been investigated. Here, we investigated the effects of pharmacological modulation of PPARα in the rat mPFC on formalin‐evoked nociceptive behaviour and the impact of formalin‐induced nociception on components of PPARα signalling in the mPFC.

Repeated forced swim stress differentially affects formalin-evoked nociceptive behaviour and the endocannabinoid system in stress normo-responsive and stress hyper-responsive rat strains.

Repeated exposure to a homotypic stressor such as forced swimming enhances nociceptive responding in rats. However, the influence of genetic background on this stress-induced hyperalgesia is poorly understood. The aim of the present study was to compare the effects of repeated forced swim stress on nociceptive responding in Sprague-Dawley (SD) rats versus the Wistar Kyoto (WKY) rat strain, a genetic background that is susceptible to stress, negative affect and hyperalgesia. Given the well-documented role of the endocannabinoid system in stress and pain, we investigated associated alterations in endocannabinoid signalling in the dorsal horn of the spinal cord and amygdala. In SD rats, repeated forced swim stress for 10 days was associated with enhanced late phase formalin-evoked nociceptive behaviour, compared with naive, non-stressed SD controls. In contrast, WKY rats exposed to 10 days of swim stress displayed reduced late phase formalin-evoked nociceptive behaviour. Swim stress increased levels of monoacylglycerol lipase (MAGL) mRNA in the ipsilateral side of the dorsal spinal cord of SD rats, an effect not observed in WKY rats. In the amygdala, swim stress reduced anandamide (AEA) levels in the contralateral amygdala of SD rats, but not WKY rats. Additional within-strain differences in levels of CB1 receptor and fatty acid amide hydrolase (FAAH) mRNA and levels of 2-arachidonylglycerol (2-AG) were observed between the ipsilateral and contralateral sides of the dorsal horn and/or amygdala. These data indicate that the effects of repeated stress on inflammatory pain-related behaviour are different in two rat strains that differ with respect to stress responsivity and affective state and implicate the endocannabinoid system in the spinal cord and amygdala in these differences.

Microinjection of 2-arachidonoyl glycerol into the rat ventral hippocampus differentially modulates contextually induced fear, depending on a persistent pain state

The endogenous cannabinoid (endocannabinoid) system plays a key role in the modulation of aversive and nociceptive behaviour. The components of the endocannabinoid system are expressed throughout the hippocampus, a brain region implicated in both conditioned fear and pain. In light of evidence that pain can impact on the expression of fear‐related behaviour, and vice versa, we hypothesised that exogenous administration of the endocannabinoid 2‐arachidonoyl glycerol (2‐AG) into the ventral hippocampus (vHip) would differentially regulate fear responding in the absence vs. the presence of formalin‐evoked nociceptive tone. Fear‐conditioned rats showed significantly increased freezing and a reduction in formalin‐evoked nociceptive behaviour upon re‐exposure to a context previously paired with footshock. Bilateral microinjection of 2‐AG into the vHip significantly reduced contextually induced freezing in non‐formalin‐treated rats, and reduced formalin‐evoked nociceptive behaviour in non‐fear‐conditioned rats. In contrast, 2‐AG microinjection had no effect on fear responding in formalin‐treated rats, and no effect on nociceptive behaviour in fear‐conditioned rats. The inhibitory effect of 2‐AG on fear‐related behaviour, but not pain‐related behaviour, was blocked by co‐administration of the cannabinoid receptor 1 (CB1) antagonist/inverse agonist rimonabant. Tissue levels of the endocannabinoids N‐arachidonoylethanolamide (anandamide, AEA) and 2‐AG were similar in the vHip of fear‐conditioned rats receiving formalin injection and the vHip of fear‐conditioned rats receiving saline injection. However, the levels of AEA and 2‐AG were significantly lower in the contralateral ventrolateral periaqueductal grey of formalin‐treated fear‐conditioned rats than in that of their saline‐treated counterparts. These data suggest that 2‐AG–CB1 receptor signalling in the vHip has an anti‐aversive effect, and that this effect is abolished in the presence of a persistent pain state.