Benchmark II kjk103

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Author: Kayla Kindig


Arguments for the Hypothesis

The pain of migraine has been suggested to be due to the sensitization of meningeal afferent neurons, since migraine patients experience a heightened sensitivity to head movements and head/facial touch during the headache phase [1][2][3]. It has been found that chemical stimulation of meningeal neurons with inflammatory mediators causes excitation and elevates their sensitivity so that they respond to stimuli previously below threshold [1]. This could explain the theory that vasodilation is capable of causing migraines, in that the trigeminal projections around the meningeal vessels are sensitized to elicit a pain response to previously innocuous stimuli, such as the expansion of blood vessels. However, the question remains as to what causes the sensitization during migraine in the first place. The established presence of CGRP in the blood during migraine[4], the ability of CGRP injection to cause migraine [5], and the ability of CGRP antagonists to relieve migraine [6][7][8] all provide basis for investigating the role of CGRP in trigeminal sensitization.


Bennett et al. (2000) tested the role of CGRP8-37, a CGRP receptor antagonist, in reducing thermal and mechanical allodynia in a rat model of central neuropathic pain. In the experiment performed by Bennett and colleagues, the spinal cords of rats were hemisected and a catheter was inserted to allow for application of CGRP8-37 after surgical recovery[9]. Spinal hemisection was chosen due to its presumed suitability as a model of chronic pain, and due to the localization of CGRP in portions of the spinal cord[9]. Their control group received a similar operation without spinal hemisection, and received injections of a vehicle peptide in order to control for potential effects of injection stress. They tested the response of the rats to stimuli mimicking a light touch, a poke, and a pin prick to the fore and hind limbs, as well as the time taken to respond to an increase in temperature. In the test group, they found that the number of withdrawals increased from the baseline established before surgery to all stimuli except the pin prick, with no significant change within the control group. They found that the highest dose of CGRP8-37 (50 nM) reduced the number of withdrawals in response to innocuous stimuli, but not the pin prick, for the experimental group, with no significant change for the control group. Responses to temperature were similar, with a decreased response time to an increase in temperature for the hemisected rats compared to baseline and controls, and the effect was alleviated by CGRP8-37. The authors established the potential pain causing action of CGRP by injecting CGRP directly, which caused the rats to writhe, increase vocalization, and bite the site of the catheter. They determined that CGRP8-37 had no direct effect on motor function by testing motor scores at each drug injection, and these scores did not differ significantly from pre-drug values.

The results obtained by Bennett and colleagues suggest that CGRP plays a role in nociception that occurs with chronic pain. They saw no change in withdrawal from noxious stimuli in controls, which provides no evidence for involvement in normal nociceptive pathways. The authors suggest this may be due to a chronic change in CGRP receptor activation following spinal injury. They propose that this increase in receptor activation could be due to an increase in neurotransmitter release. Interestingly, they explain that CGRP distribution in the spinal cord increases following spinal hemisection, thus suggesting an increase in the number of CGRP-containing synaptic terminals. It is not clear from this data what the mechanism of action of is for CGRP, or even if it is pre- or postsynaptic. The authors propose an effect on other neurotransmitters, as CGRP has been shown to prolong the action of substance P [9], and stimulate the release of excitatory amino acids aspartate and glutamate [10]. Glutamate acts on NMDA receptors, and NMDA receptors play a role in synaptic plasticity [11]. Thus, CGRP could cause sensitization via the activation of NMDA, which could act to lower the nociceptive threshold of trigeminal neurons. Additionally, substance P is a peptide that has been linked to nociception[12]. CGRP is thought to prolong the action of substance P by preventing its enzymatic breakdown, due to the two compounds sharing the same degradative enzymes[13]. If there was an excess of CGRP, the enzymes would be more likely to degrade CGRP and substance P could remain in the synaptic cleft and act for a longer time. Alternatively, there is also evidence that CGRP can elicit pain transmission independently of substance P or glutamate, in that CGRP alone is capable of eliciting a depolarization in spinal cord dorsal horn neurons [14].

Although the results are consistent with CGRP being involved in sensitization, the behavior of CGRP in the central nervous system during migraine may differ from that in the spinal cord. Additionally, spinal injury was required for the relief of nociception by CGRP8-37, and while this is a general condition designed to evoke the sensitization of neurons to non-painful stimuli, it is unclear if the results are completely applicable to migraine, which presumably requires no physical injury to occur.


Sun et al. (2007) further investigated the role of CGRP in neuronal sensitization. In their study, rats were anesthetized, and a catheter was inserted into subarachnoid space[15]. The rats were then paralyzed and the spinal cord exposed. For behavioral experiments, the experimenters injected the drugs, and for the electrophysiological experiments, the drugs were directly applied to the spinal cord. To assess behavioral changes to stimuli, they applied von Frey filaments of logarithmically increasing stiffnesses for 6-8 seconds and recorded if the paw of the animal was withdrawn. To record electrophysiological data, they recorded responses of wide dynamic range (WDR) neurons in the spinal cord that respond best to noxious stimuli. They performed extracellular recordings of WDR neurons in the L5 segment of the spinal cord, which all had receptive fields on the plantar surface of the hindpaw, determined by brush and pinch stimuli. The authors found that injection of CGRP reduced the threshold for paw withdrawal to von Frey filaments of about half the stiffness as before injection of CGRP or controls injected with DMSO. The effect was mitigated by addition of chelerythrine chloride (CC), which inhibits protein kinase C (PKC). The effect was also mitigated by application of chelerythrine (H89), which inhibits protein kinase A (PKA). They found that infusion of CGRP increased the firing rate of WDR neurons by about 50% of baseline in response to a press stimulus. They found no significant increase in firing rate from background or baseline levels when infused with CC or H89 30 minutes before infusion of CGRP.

The results of this study further demonstrate that CGRP is involved in sensitization of neurons required to reduce the pain threshold. The authors demonstrate this sensitization both with behavioral and electrophysiological data. Additionally, this study provides a potential mechanism by which this occurs: activation of PKC and PKA. It has been demonstrated that the CGRP receptor activates cAMP as a second messenger, which leads to downstream activation of PKA [16]. Due to the limitations of the study by Sun et al., it cannot be determined in what order PKC and PKA pathways are activated, but it is possible that they function together. PKA and PKC both have supported roles in neuron sensitization [17][18]. Protein kinase C specifically has been demonstrated to enhance NMDA-evoked currents [19], possibly by regulation of receptor gating [20]. There is evidence that NMDA EPSCs are dependent on 5-HT2A receptor activation, but only when PKC is active [21]. This suggests a mechanism in which 5-HT2A receptor activation leads to activation of PKC, which causes the phosphorylation of NMDA receptor subunits and consequent upregulation of NMDA [21]. Since NMDA affects synaptic plasticity, it is possible that this may lead to the enhanced excitation of nociceptive neurons. This mechanism could be able to explain why serotonin receptor agonists like Sumatriptan that are used to treat migraine headache can be ineffective and cause rebound headaches; although the primary function of triptan derivatives is to cause vasoconstriction, they may be inadvertently leading to neuronal excitation.


Gu and Yu (2007) investigated the co-localization of CGRP with AMPA receptors in the spinal cord. The authors used double immunofluorescence labeling to localize CGRP and AMPA receptors in the dorsal horn of the spinal cord in rats[22]. The spinal cord was fixed, transected, and incubated with antibodies against a sequence at the C-terminal of the human CGRP receptor and an antibody against the GluR2 subunit of AMPA receptors. They found that there was significant overlap between the localization of CGRP receptors and AMPA receptors in the soma of dorsal horn neurons. They then performed a single-cell extracellular recording to confirm that their similar localization indicated a similar function. They exposed the L2-L4 dorsal surface of the spinal cord via laminectomy of anesthetized rats, and used varying grades of stimulation to determine the receptive field of the neuron. They recorded the average frequency per minute of discharge from a WDR neuron. They found that application of both CGRP and AMPA increased the average discharge frequency of the neuron by up to 75% from baseline.

The results of this study further provide evidence for the sensitization of neurons by CGRP, and also gives insight into a potential mechanism. AMPA receptors respond to glutamate, an excitatory neurotransmitter, and has been implicated in the transmission of sensory information[22]. Both AMPA and CGRP increased the firing frequency of WDR neurons in the spinal dorsal horn, and both are localized to similar areas. However, this study does not provide any insight as to whether the site of action of CGRP is pre- or postsynaptic, since the drug is free to diffuse after application. Additionally, this study does not provide direct evidence for an interaction between AMPA and CGRP, but the functional and localization similarity is indirect evidence. It should be noted that since the authors did not apply a CGRP antagonist to see if that was able to stop the increased responsivity, it is unclear by looking at this study if CGRP is sufficient to cause the effects. However, taken with the results of other studies where CGRP antagonist did mitigate the effects, it can be presumed that CGRP is necessary. Hypothetically, if CGRP increases the quantitiy of AMPA receptors on nociceptive neurons, this could lead to the perception of pain by making the synapses of nociceptive neurons more excitable, as increased levels of AMPA receptors can alter protein synthesis over time and lead to synaptic remodeling [11]. This could occur by a similar pathway as described for PKC and NMDA receptors, in that CGRP receptor activation could initiate a cascade involving the phosphorylation of AMPA receptors by PKC or PKA.


Adwaninkar et al. (2007) investigated the role of CGRP in the nociceptive signaling pathway of the amygdala. They induced arthritis in the knee of rats by injection of kaolin suspension and carrageenan [23]. They performed single-cell extracellular recordings on amygdala neurons of the latero-capsular division of the central nucleus (CeLC). They stimulated the neurons by applying mechanical stimuli, delivered by forceps, to the receptive field, which included the knee and the ankle. They defined stimuli as innocuous (100 and 500 g/30mm^2) or noxious (1500 and 2000 g/30mm^2) based on whether it evoked the withdrawal reflex in conscious mice and if it was painful to experimenters. They chose to test neurons that respond more strongly to noxious stimuli than innocuous stimuli, and called them multireceptive (MR) neurons. They recorded neuron response to stimuli before and 6 hours after induction of arthritis in the knee. They applied the CGRP receptor antagonists CGRP8-37 and BIBN4096BS to control rats with no induced arthritis, and to arthritic rats 5-6 hours after injection. They applied mechanical stimulus to the hindpaw of rats and determined the threshold required for paw withdrawal, and recorded vocalizations made by the rats during stimulation. They found that firing rate was greater in arthritic mice for both innocuous and noxious stimuli, but was reduced by application of both CGRP8-37 and BIBN4096BS, and increased again after washout of the drugs. They found no effect of CGRP8-37 or BIBN4096BS on normal mice. Vocalization in both the audible and ultrasonic range was longer in duration for arthritic mice, but was decreased with application of both CGRP8-37 and BIBN4096BS. They found that the threshold for hindpaw withdrawal was lowered in arthritic mice, from about 1,000 g/30mm^2 in regular mice to ~600 g/30mm^2 in arthritic mice. This effect was reversed by CGRP8-37 and BIBN4096BS, restoring the threshold to approximately 1,0000 g/30mm^2.

This study provides further support for the role of CCGRP in sensitization of neurons. The fact that firing rate increased again in arthritic mice after washout of the CGRP receptor antagonist is compelling evidence, since it suggests the decrease in firing rate is not merely to the passage of time, but is a direct consequence of the CGRP antagonists. Again, the results of this study suggest that CGRP is not involved in normal perception of sensory information, but in chronic pain sensitization. This study also provides evidence of involvement of CGRP with a brain structure associated with responding to nociceptive information [23]. The amygdala has been implicated in emotional responses that are associated with pain, such as fear [24] and anxiety [25]. It is perhaps worth noting that fear and anxiety are components of stress and/or causes of stress, and stress is a key trigger of migraine headaches for many patients [26]. This could potentially explain why migraine patients are able to feel the headache pain without the presence of an external pain stimulus, in that feelings associated with pain could have the ability to initiate or mediate a response.


The studies examined here all point to a mechanism involving CGRP receptors on neurons, but it is important to note that the CGRP receptor is found in multiple structures of the brain as well as other cell types, and it may act differently depending on which cell type is being examined. For example, CGRP receptors have also been identified in the satelite glia as well as neurons of the trigeminal ganglion [27]. One important mechanism to consider is the involvement of CGRP receptors that reside on the smooth muscle of dural blood vessels. CGRP has been established as a potent vasodilator [28], and a pervasive theory as to what causes the sensitization of neurons is a sterile inflammatory response, in which mast cell degranulation may alter the sensitivity of nociceptors in the meninges[29]. CGRP may evoke sensitization through its action as a vasodilator by triggering an inflammatory response. It has been shown that CGRP and substance P application lead to the release of histamine from dural mast cells but not peritoneal mast cells, an effect that is reversed by applying a CGRP receptor antagonist[30]. This suggests a mechanism in which CGRP is released from the trigeminal neurons to both dilate dural blood vessels and stimulate mast cells to release histamine, and histamine is both taken up by the neuron and is diffused into the blood vessel to allow for inflammatory plasma extravasation.


References

  1. ↑ 1.0 1.1 Strassman, A.; Raymond, S.; Burstein, R. (1996). "Sensitization of meningeal sensory neurons and the origin of headaches". Nature. 384: 560–564. 
  2. ↑ Wiesler, J.; Ellis, A.; Sprunger, D.; Brown, K.; McFadden, A.; Mahoney, J.; Watkins, L.R. (2010). "A novel method for modeling facial allodynia associated with migraine in awake and freely moving rats". Journal of Neuroscience Methods. 185 (2): 236. 
  3. ↑ Burstein, R.; Cutrer, M.; Yarnitsky, D. (2000). "The development of cutaneous allodynia during a migraine attack". Brain. 123: 1703–1709. 
  4. ↑ Goadsby, P.J.; Edvinsson, L.; Ekman, R. (1990). "Vasoactive peptide release in the extracerebral circulation of humans during migraine attacks.". Annals of Neurology. 28: 183–187. 
  5. ↑ Lassen, L.; Haderslev, P.; Jacobsen, V.; Iversen, H.; Sperling, B.; Olesen, J. (2002). "CGRP may play a causative role in migraine.". Cephalalgia. 22: 54–61. 
  6. ↑ Verheggen, R.; Bumann, K.; Kaumann, A. (2002). "BIBN4096BS is a potent competitive antagonist of the relaxant effects of alpha-CGRP on human temporal artery: comparison with CGRP(8-37)". British Journal of Pharmacology. 136: 120–126. 
  7. ↑ Bigal, M.; Walter, S. (2014). "Monoclonal antibodies for migraine: preventing calcitonin gene-related peptide activity". Central Nervous System Drugs. 28: 389–399. 
  8. ↑ Ho, T.; Mannix, L.; Fan, X.; Assaid, C.; Furtek, C.; Jones, J.; Lines., C.; Rapoport, A. (2008). "Randomized controlled trial of an oral CGRP receptor antagonist, MK-0974, in acute treatment of migraine". Neurology. 70 (16): 1304–1312. 
  9. ↑ 9.0 9.1 9.2 Bennett, A.; Chastain, K.; Hulsebosch, C. (2000). "Alleviation of mechanical and thermal allodynia by CGRP8-37 in a rodent model of chronic central pain". Pain. 86 (1-2): 163–175. 
  10. ↑ Kangra, I.; Larew, J.; Randic, M. (1990). "The effects of substance P and calcitonin gene-related peptide on the ef¯ux of endogenous glutamate and aspartate from the rat spinal dorsal horn in vitro". Neuroscience Letters. 108: 155–160. 
  11. ↑ 11.0 11.1 Chiel, Hillel. "Synaptic Plasticity II: Introduction to Long-Term Potentiation". Neurowiki. Retrieved 4 November 2016. 
  12. ↑ Chiel, Hillel. "Novel Transmitters I: Introduction to Nitric Oxide". Neurowiki. Retrieved 4 November 2016. 
  13. ↑ Chen, J.; Barber, L.; Dymshitz, J.; Vaski, M. (1996). "Peptidase inhibitors improve recovery of substance P and calcitonin gene-related peptide release from rat spinal cord slices". Peptides. 17: 31–37. 
  14. ↑ Miletic, V.; Tan, H. (1988). "Iontophoretic application of calcitonin gene-related peptide produces a slow and prolonged excitation of neurons in the cat lumber dorsal horn.". Brain Research. 446: 169–172. 
  15. ↑ Sun, Q.; Tu, Y.; Lawand, B.; Yan, J.; Lin, Q.; Willis, W. (2004). "Calcitonin gene-related peptide receptor activation produces PKA- and PKC-dependent mechanical hyperalgesia and central sensitization". Journal of Neurophysiology. 92: 2859–2866. 
  16. ↑ Poyner, D. (1992). "Calcitonin gene-related peptide: multiple actions, multiple receptors". Pharmacology Therapy. 56: 32–51. 
  17. ↑ Lin, Q.; Peng, Y.; Willis, W. (1996). "Possible role of protein kinase C in the sensitization of primate spinothalamic tract neurons.". Journal of Neuroscience. 16: 3026–3034. 
  18. ↑ Lin, Q.; Willis, W.; Wu, J. (2002). "Effect of protein kinase A activation on the responses of primate spinothalamic tract neurons to mechanical stimuli". Journal of Neurophysiology. 88: 214–221. 
  19. ↑ Lu., W.; Xiong, S.; Lei, S.; Orser, B.; Dudek, E.; Browning, M.; MacDonald, J. (1999). "G-protein-coupled receptors act via protein kinase C and Src to regulate NMDA receptors". Nature Neuroscience. 2: 331338. 
  20. ↑ Lin, Y.; Jover-Mengual, T.; Wong, J.; Bennett, M.; Zukin, R. (2006). "PSD-95 and PKC converge in regulating NMDA receptor trafficking and gating". Proceedings of the National Academy of Sciences. 103 (52): 19902–19907. 
  21. ↑ 21.0 21.1 Barre, A.; Berthoux, C.; Bundel, D.; Valjent, E.; Bockaert, J.; Marin, P.; Becamel, C. (2016). "Presynaptic serotonin 2A receptors modulate thalamocortical plasticity and associative learning". Proceedings of the National Academy of Sciences. 113 (10): E1382–E1391. 
  22. ↑ 22.0 22.1 Gu, X.; Yu, L. (2007). "The colocalization of CGRP receptor and AMPA receptor in the spinal dorsal horn neuron of rat: A morphological and electrophysiological study". Neuroscience Letters. 414: 237–241. 
  23. ↑ 23.0 23.1 Adwanikar, H.; Ji, G.; Li, W.; Doods, H.; Willis, W.; Neugebauer, V. (2007). "Spinal CGRP1 receptors contribute to supraspinally organized pain behavior and pain-related sensitization of amygdala neurons". Pain. 132 (1-2): 53–66. 
  24. ↑ Feinstein, J.; Adolphs, R.; Damasio, A.; Tranel, D. (2011). "The human amygdala and the induction and experience of fear". Current Biology. 21 (1): 34–38. 
  25. ↑ Ziabreva, I.; Poeggel, G.; Schnabel, R.; Braun, K. (2003). "Separation-Induced Receptor Changes in the Hippocampus and Amygdala of Octodon degus: Influence of Maternal Vocalizations". Journal of Neuroscience. 23 (12): 5329–5336. 
  26. ↑ "NINDS Migraine Information Page". National Institute of Neurological Disorders and Stroke. National Institutes of Health. Retrieved 20 October 2016. 
  27. ↑ Durham, P.; Vause, C. (2010). "CGRP receptor antagonists in the treatment of migraine". Central Nervous System Drugs. 24 (7): 539–548. 
  28. ↑ McCulloch, J; Uddman, R.; Kingman, T.; Edvinsson, L. (1986). "Calcitonin gene-related peptide: Functional role in cerebrovascular regulation". Proceedings of the National Academy of Sciences USA. 83: 5731–5735. 
  29. ↑ Zhang, X.; Strassman, A.; Burstein, R.; Levy, D. (2007). "Sensitization and activation of intracranial meningeal nociceptors by mast cell mediators". The Journal of Pharmacology and Experimental Therapeutics. 322 (2): 806–812. 
  30. ↑ Ottosson, A.; Edvinsson, L. (1997). "Release of histamine from dural mast cells by substance P and calcitonin gene-related peptide". Cephalalgia. 17: 166–174. 
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