Nociception assay

Last updated April 29, 2024

A nociception assay (nocioception or nocioperception assay) evaluates the ability of an animal, usually a rodent, to detect a noxious stimulus such as the feeling of pain, caused by stimulation of nociceptors. These assays measure the existence of pain through behaviors such as withdrawal, licking, immobility, and vocalization. The sensation of pain is not a unitary concept; therefore, a researcher must be conscious as to which nociception assay to use.

Formalin

The formalin assay is the most popular chemical assay of nociception. It entails the injection of a dilute solution of formalin into the surface of the rodent's hindpaw, followed by the scoring of stereotypical behaviors such as flinching, licking, and biting of the affected hindpaw.^[1] The behaviors last for approximately 1 hour, with the early or acute stage (directly after injection) reflecting direct activation of nociceptors and the late or tonic phase (15 to 20 minutes after the injection) reflecting inflammation.^[1] Typically, the formalin assay is used on rats; however, formalin concentrations and scoring methods can be modified as to suit mice.^[2] One major advantage of the formalin assay over other models of inflammatory pain is the limited duration (approximately 1 hour) of the response.^[2] Additionally, as described before, this assay produces a response in two discrete stages, allowing researchers to model both acute and tonic pain using a single noxious chemical.

Writhing

In the writhing test, the peripheral nociceptive activity of a test compound is determined by the number of abdominal writhes induced by the intraperitoneal injection of acetic acid.^[3]^[4]

Von Frey

The Von Frey assay, introduced by Maximilian von Frey and modified by Weinstein, uses Von Frey hair or fibers, which are small pieces of nylon rod, approximately 50 mm in length, and of varying diameters, to test a rodent's sensitivity to a mechanical stimulus.^[1] It is unclear whether the process is really considered noxious versus simply annoying, so this assay is a test of mechanical nociception or simply mechanical sensibility. In this test, the animal stands on an elevated mesh platform, and the Von Frey hairs are inserted through the mesh to poke the animal’s hindpaw.^[2] Normal reactions for the animal include withdrawing or licking or shaking the paw, and possible vocalization, but these can depend on variability within the experiment. For example, the tarsal surface of the hind paw is typically associated with lower withdrawal thresholds compared to the dorsal surface, and the exact force of the fiber is determined by its thickness.^[2] It is also important to note that thresholds usually are initially decreasing during successive tests, but do become stable after about 3 sessions. Algorithms such as up-down or Bruceton analysis are available to concentrate testing into the most dynamic part of the range, and subsequent curve fitting and parameter estimation can be similarly standardised.^[5] Alternatively, automated von Frey systems have recently been discovered that gradually increase the force of a single probe so that a researcher can observe when withdrawal responses occur.

Thermal assays

Sensitivity to acute thermal stimulation is the most common test used in live species pain research.^[2] The behavioral reflex evoked by noxious heat stimuli is a relatively good predictor of pain sensitivity and its reduction through various analgesics. One significant limitation of thermal assays lies in the specificity and validity of results in animals as models of human pain.^[2] Very little is known about the functional mechanics of nociceptive afferents in murine subjects, thus the translation of any pain response observed from these animals to humans is questionable.^[2]

Tail withdrawal

Two versions of the tail withdrawal assay are commonly employed in pain sensitivity testing.^[2] In the classic radiant heat test, a heat source is targeted onto a small area of the tail, and the latency to withdraw the tail away from the heat source is measured. In the tail-immersion test, a container of liquid is heated or cooled to a nociceptive temperature – normally 50–55 °C or below 0 °C. The animal subject is then placed with its tail immersed in the liquid, and the latency to withdraw the tail from the liquid is measured.

Animal subjects used must be restrained to a fairly high degree when performing the tail withdrawal test due to the exact positioning necessary to direct the noxious stimuli. Restraint is usually accomplished by placing the subjects in small Plexiglas tubes or cloth/cardboard pockets that the subjects can either be habituated to or voluntarily enter.^[2]

The primary advantage of tail withdrawal assays to other forms of thermal nociception testing, such as the hot-plate test or Hargreaves test, is the relative stability of results with repeated observations. Pain-reflex latency observations from other tests are usually much more variable both across and within subjects than those obtained from the tail withdrawal assay.

Hot plate

A heat-conductive surface, such as porcelain or metal, is heated to a temperature that will induce a nociceptive response in an animal subject – normally 50–56 °C.^[2] The subject is then placed onto the surface and prevented from leaving the platform by blockades. The latency to pain-reflex behavior is measured.^[1] One complication of this assay is its unsuitability for repeated testing. Animals that have been subjected to the hot-plate test in the past display a behavioral tolerance phenomenon, which is characterized by decreased latencies and reduced sensitivities to antinociceptive agents.^[1] Another complication of the hot-plate test is determining what constitutes a behavioral pain response; is it the lifting/licking of paws, vocalization, attempting to climb out of the cylinder, etc.^[2] Also, delivering the heat stimulus in a controlled fashion presents difficulties due to each section having varying temperatures based upon surface area exposure and whether the animal is moving or not.^[2]

Tail flick

The tail flick assay or tail flick test uses a high-intensity beam of light aimed at a rodent's tail to detect nociception.^[1] In normal rodents, the noxious heat sensation induced by the beam of light causes a prototypical movement of the tail via the flexor withdrawal reflex.^[2] An investigator normally measures the time it takes for the reflex to be induced, a factor influenced by a rodent's sex, age and body weight.^[1] The most critical parameter for the tail flick assay is the beam intensity; stimuli producing latencies of larger than 3–4 seconds generally create more variable results.^[6] Another important factor to consider is the level of restraint used; rodents held too tightly may exhibit greater tail flick latencies due to heightened stress levels.^[6]

Hargreaves

The Hargreaves assay uses a high-intensity beam of light directed at the hindpaw rather than the tail to induce pain; an investigator then measures the time it takes for the animal to withdraw its hindpaw.^[1] In contrast to the tail flick assay, rodents are often unrestrained while the radiant heat source is focused on the hindpaw. Cut-off latency for the Hargreaves assay is commonly set at 10 seconds.^[7] The main advantage of this test over the tail flick assay is that it allows independent assessment of treatment effects on both sides of the body.^[2]

Applications

One of the most common applications of nociception assays is to test the effectiveness of new pain medications and drugs of the like. One can then perform comparative tests to measure the differences in the effects of the drug on varying populations, such as men versus women, or young versus old. These tests can also identify certain harmful diseases or abnormalities in subjects if they display atypical nociception test responses. Additionally, nociception tests can be used to test the heritability of nociception itself.^[8] One can also use nociception assays to assess the physiology of the "pain" pathways. The role capsaicin receptors play in the pain pathways has been measured by comparing results from nociception assays in mice with and without the receptor.^[9] In addition, they are useful in other tests to make sure control subjects have normal nociception responses.

Related Research Articles

In physiology, nociception, also nocioception; from Latin nocere 'to harm/hurt') is the sensory nervous system's process of encoding noxious stimuli. It deals with a series of events and processes required for an organism to receive a painful stimulus, convert it to a molecular signal, and recognize and characterize the signal to trigger an appropriate defensive response.

A nociceptor is a sensory neuron that responds to damaging or potentially damaging stimuli by sending "possible threat" signals to the spinal cord and the brain. The brain creates the sensation of pain to direct attention to the body part, so the threat can be mitigated; this process is called nociception.

<span class="mw-page-title-main">Nociceptin</span> Chemical compound

Nociceptin/orphanin FQ (N/OFQ), a 17-amino acid neuropeptide, is the endogenous ligand for the nociceptin receptor. Nociceptin acts as a potent anti-analgesic, effectively counteracting the effect of pain-relievers; its activation is associated with brain functions such as pain sensation and fear learning.

The ventrobasal complex (VB) is a relay nucleus of the thalamus for nociceptive stimuli received from nociceptive nerves. The VB consists of the ventral posteromedial nucleus (VPM) and the ventral posterolateral nucleus (VPL). In some species, the ventral posterolateral nucleus, pars caudalis is also a part of the VB. The VB gets inputs from the spinothalamic tract, medial lemniscus, and corticothalamic tract. The main output of the VB is the primary somatosensory cortex.

Neuromedin U is a neuropeptide found in the brain of humans and other mammals, which has a number of diverse functions including contraction of smooth muscle, regulation of blood pressure, pain perception, appetite, bone growth, and hormone release. It was first isolated from the spinal cord in 1985, and named after its ability to cause smooth muscle contraction in the uterus.

Transient receptor potential cation channel, subfamily A, member 1, also known as transient receptor potential ankyrin 1, TRPA1, or The Wasabi Receptor, is a protein that in humans is encoded by the TRPA1 gene.

A noxious stimulus is a stimulus strong enough to threaten the body's integrity. Noxious stimulation induces peripheral afferents responsible for transducing pain throughout the nervous system of an organism.

Fish fulfill several criteria proposed as indicating that non-human animals experience pain. These fulfilled criteria include a suitable nervous system and sensory receptors, opioid receptors and reduced responses to noxious stimuli when given analgesics and local anaesthetics, physiological changes to noxious stimuli, displaying protective motor reactions, exhibiting avoidance learning and making trade-offs between noxious stimulus avoidance and other motivational requirements.

Pain negatively affects the health and welfare of animals. "Pain" is defined by the International Association for the Study of Pain as "an unpleasant sensory and emotional experience associated with actual or potential tissue damage, or described in terms of such damage." Only the animal experiencing the pain can know the pain's quality and intensity, and the degree of suffering. It is harder, if even possible, for an observer to know whether an emotional experience has occurred, especially if the sufferer cannot communicate. Therefore, this concept is often excluded in definitions of pain in animals, such as that provided by Zimmerman: "an aversive sensory experience caused by actual or potential injury that elicits protective motor and vegetative reactions, results in learned avoidance and may modify species-specific behaviour, including social behaviour." Nonhuman animals cannot report their feelings to language-using humans in the same manner as human communication, but observation of their behaviour provides a reasonable indication as to the extent of their pain. Just as with doctors and medics who sometimes share no common language with their patients, the indicators of pain can still be understood.

There is a scientific debate which questions whether crustaceans experience pain. It is a complex mental state, with a distinct perceptual quality but also associated with suffering, which is an emotional state. Because of this complexity, the presence of pain in an animal, or another human for that matter, cannot be determined unambiguously using observational methods, but the conclusion that animals experience pain is often inferred on the basis of likely presence of phenomenal consciousness which is deduced from comparative brain physiology as well as physical and behavioural reactions.

The tail flick test is a test of the pain response in animals, similar to the hot plate test. It is used in basic pain research and to measure the effectiveness of analgesics, by observing the reaction to heat. It was first described by D'Amour and Smith in 1941.

The hot plate test is a test of the pain response in animals, similar to the tail flick test. Both hot plate and tail-flick methods are used generally for centrally acting analgesic, while peripherally acting drugs are ineffective in these tests but sensitive to acetic acid-induced writhing test.

Pain in invertebrates is a contentious issue. Although there are numerous definitions of pain, almost all involve two key components. First, nociception is required. This is the ability to detect noxious stimuli which evokes a reflex response that moves the entire animal, or the affected part of its body, away from the source of the stimulus. The concept of nociception does not necessarily imply any adverse, subjective feeling; it is a reflex action. The second component is the experience of "pain" itself, or suffering—i.e., the internal, emotional interpretation of the nociceptive experience. Pain is therefore a private, emotional experience. Pain cannot be directly measured in other animals, including other humans; responses to putatively painful stimuli can be measured, but not the experience itself. To address this problem when assessing the capacity of other species to experience pain, argument-by-analogy is used. This is based on the principle that if a non-human animal's responses to stimuli are similar to those of humans, it is likely to have had an analogous experience. It has been argued that if a pin is stuck in a chimpanzee's finger and they rapidly withdraw their hand, then argument-by-analogy implies that like humans, they felt pain. It has been questioned why the inference does not then follow that a cockroach experiences pain when it writhes after being stuck with a pin. This argument-by-analogy approach to the concept of pain in invertebrates has been followed by others.

<span class="mw-page-title-main">Rostral ventromedial medulla</span> Group of neurons in medulla of brain

The rostral ventromedial medulla (RVM), or ventromedial nucleus of the spinal cord, is a group of neurons located close to the midline on the floor of the medulla oblongata. The rostral ventromedial medulla sends descending inhibitory and excitatory fibers to the dorsal horn spinal cord neurons. There are 3 categories of neurons in the RVM: on-cells, off-cells, and neutral cells. They are characterized by their response to nociceptive input. Off-cells show a transitory decrease in firing rate right before a nociceptive reflex, and are theorized to be inhibitory. Activation of off-cells, either by morphine or by any other means, results in antinociception. On-cells show a burst of activity immediately preceding nociceptive input, and are theorized to be contributing to the excitatory drive. Neutral cells show no response to nociceptive input.

Maria Carmela Lico or Licco (1927–1985) spent most of her research life as a physiologist studying the neural mechanisms of pain at the Department of Physiology of the Faculdade de Medicina de Ribeirão Preto (Brazil). Lico produced important insights on the descending control of nociception by limbic structures, specially the septal nuclei.

Pain is an aversive sensation and feeling associated with actual, or potential, tissue damage. It is widely accepted by a broad spectrum of scientists and philosophers that non-human animals can perceive pain, including pain in amphibians.

Pain in cephalopods is a contentious issue. Pain is a complex mental state, with a distinct perceptual quality but also associated with suffering, which is an emotional state. Because of this complexity, the presence of pain in non-human animals, or another human for that matter, cannot be determined unambiguously using observational methods, but the conclusion that animals experience pain is often inferred on the basis of likely presence of phenomenal consciousness which is deduced from comparative brain physiology as well as physical and behavioural reactions.

<span class="mw-page-title-main">RB-120</span> Chemical compound

RB-120 is an orally active analog of the drug RB-101. It acts as an enkephalinase inhibitor, which is used in scientific research. Via intravenous administration, it is approximately three times as potent as RB-101 or twice as potent as the isolated (S,S) isomer of RB101. However, via i.p. administration it is approximately twice as potent as racemic RB-101 and about as potent as the isolated (S,S) isomer of RB101. During i.v. administration RB120 is approximately twice as weak as morphine in terms of analgesia; however, it is 16x weaker during i.p. and p.o. administration.

<span class="mw-page-title-main">Mitragynine</span> Opioid analgesic compound

Mitragynine is an indole-based alkaloid and the most abundant active alkaloid in the Southeast Asian plant Mitragyna speciosa, commonly known as kratom. The total alkaloid concentration in dried leaves ranges from 0.5 to 1.5%. In Thai varieties, mitragynine is the most abundant component while 7-hydroxymitragynine is a minor constituent. In Malaysian kratom varieties, mitragynine is present at lower concentration. Such preparations are orally consumed and typically involve dried kratom leaves which are brewed into tea or ground and placed into capsules. Mitragynine consumption for medicinal and recreation purposes dates back centuries, although early use was primarily limited to Southeast Asian countries such as Indonesia and Thailand where the plant grows indigenously. Recently, mitragynine use has spread throughout Europe and the Americas as both a recreational and medicinal drug. While research into the effects of kratom have begun to emerge, investigations on the active compound mitragynine are less common.

Sandra M. Garraway is a Canadian-American neuroscientist and assistant professor of physiology in the Department of Physiology at Emory University School of Medicine in Atlanta, Georgia. Garraway is the director of the Emory Multiplex Immunoassay Core (EMIC) where she assists researchers from both academia and industry to perform, analyze, and interpret their multiplexed immunoassays. Garraway studies the neural mechanisms of spinal nociceptive pain after spinal cord injury and as a postdoctoral researcher she discovered roles for both BDNF and ERK2 in pain sensitization and developed novel siRNA technology to inhibit ERK2 as a treatment for pain.

References

1 2 3 4 5 6 7 8 Carter, Matt; Shieh, Jennifer C. (2010). "Nociception". Guide to Research Techniques in Neuroscience. Burlington, MA: Academic Press. pp. 51–2. ISBN 978-0-12-374849-2.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 Mogil, Jeffrey; Wilson, Sonya; Wan, You (2001). "Assessing Nociception in Murine Subjects". Methods in Pain Research. Frontiers in Neuroscience. Vol. 20012652. doi:10.1201/9781420042566-c2. ISBN 978-0-8493-0035-6.
↑ Koster, R.; Anderson, M.; De Beer, J. (1959). "Acetic acid for analgesic screening". Federation Proceedings. 18: 412–417.
↑ Matera, Carlo; Flammini, Lisa; Quadri, Marta; Vivo, Valentina; Ballabeni, Vigilio; Holzgrabe, Ulrike; Mohr, Klaus; De Amici, Marco; Barocelli, Elisabetta; Bertoni, Simona; Dallanoce, Clelia (2014). "Bis(ammonio)alkane-type agonists of muscarinic acetylcholine receptors: Synthesis, in vitro functional characterization, and in vivo evaluation of their analgesic activity". European Journal of Medicinal Chemistry. 75: 222–232. doi:10.1016/j.ejmech.2014.01.032. ISSN 0223-5234. PMID 24534538.
↑ Bradman, Matthew J.G.; Ferrini, Francesco; Salio, Chiara; Merighi, Adalberto (November 2015). "Practical mechanical threshold estimation in rodents using von Frey hairs/Semmes–Weinstein monofilaments: Towards a rational method". Journal of Neuroscience Methods. 255: 92–103. doi:10.1016/j.jneumeth.2015.08.010. PMID 26296284. S2CID 206270382.
1 2 Bannon, Anthony W.; Malmberg, Annika B. (2007). Models of Nociception: Hot-Plate, Tail-Flick, and Formalin Tests in Rodents. Vol. Chapter 8. pp. Unit 8.9. doi:10.1002/0471142301.ns0809s41. ISBN 978-0-471-14230-0. PMID 18428666. S2CID 19332207.{{cite book}}: |journal= ignored (help)
↑ Varnado-Rhodes, Y; Gunther, J; Terman, GW; Chavkin, C (2000). "Mu opioid analgesia and analgesic tolerance in two mouse strains: C57BL/6 and 129/SvJ". Proceedings of the Western Pharmacology Society. 43: 15–7. PMID 11056944.
↑ Lariviere, William R; Wilson, Sonya G; Laughlin, Tinna M; Kokayeff, Anna; West, Erin E; Adhikari, Seetal M; Wan, You; Mogil, Jeffrey S (2002). "Heritability of nociception. III. Genetic relationships among commonly used assays of nociception and hypersensitivity". Pain. 97 (1–2): 75–86. doi:10.1016/S0304-3959(01)00492-4. PMID 12031781. S2CID 17419719.
↑ Caterina, M. J.; Leffler, A; Malmberg, AB; Martin, WJ; Trafton, J; Petersen-Zeitz, KR; Koltzenburg, M; Basbaum, AI; Julius, D (2000). "Impaired Nociception and Pain Sensation in Mice Lacking the Capsaicin Receptor". Science. 288 (5464): 306–13. Bibcode:2000Sci...288..306C. doi:10.1126/science.288.5464.306. PMID 10764638.

External links

Lariviere, William R; Wilson, Sonya G; Laughlin, Tinna M; Kokayeff, Anna; West, Erin E; Adhikari, Seetal M; Wan, You; Mogil, Jeffrey S (2002). "Heritability of nociception. III. Genetic relationships among commonly used assays of nociception and hypersensitivity". Pain. 97 (1–2): 75–86. doi:10.1016/S0304-3959(01)00492-4. PMID 12031781. S2CID 17419719.
Le Bars, Daniel; Gozariu, Manuela; Cadden, Samuel W. (2001). "Animal Models of Nociception". Pharmacological Reviews. 53 (4): 597–652. PMID 11734620.
Wilson, Sonya G.; Mogil, Jeffrey S. (2001). "Measuring pain in the (knockout) mouse: Big challenges in a small mammal". Behavioural Brain Research. 125 (1–2): 65–73. doi:10.1016/S0166-4328(01)00281-9. PMID 11682095. S2CID 2556160.
Rao, Tadimeti S.; Correa, Lucia D.; Reid, Richard T.; Lloyd, G.Kenneth (1996). "Evaluation of anti-nociceptive effects of neuronal nicotinic acetylcholine receptor (NAChR) ligands in the rat tail-flick assay". Neuropharmacology. 35 (4): 393–405. doi:10.1016/0028-3908(96)00013-5. PMID 8793901. S2CID 20153440.
Scheuren, N.; Neupert, W.; Ionac, M.; Neuhuber, W.; Brune, K.; Geisslinger, G. (1997). "Peripheral noxious stimulation releases spinal PGE2 during the first phase in the formalin assay of the rat". Life Sciences. 60 (21): 295–300. doi:10.1016/S0024-3205(97)00155-0. PMID 9155004.
Johansson, R.S.; Vallbo, Å.B.; Westling, G. (1980). "Thresholds of mechanosensitive afferents in the human hand as measured with von Frey hairs". Brain Research. 184 (2): 343–51. CiteSeerX 10.1.1.572.181 . doi:10.1016/0006-8993(80)90803-3. PMID 7353160. S2CID 37354867.
Lecci, Alessandro; Giuliani, Sandro; Patacchini, Riccardo; Viti, Giovanni; Maggi, Carlo Alberto (1991). "Role of NK1 tachykinin receptors in thermonociception: Effect of (±)-CP 96,345, a non-peptide substance P antagonist, on the hot plate test in mice". Neuroscience Letters. 129 (2): 299–302. doi:10.1016/0304-3940(91)90485-C. PMID 1720881. S2CID 20529870.

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[multiple-1] 1 2 3 4 5 6 7 8 Carter, Matt; Shieh, Jennifer C. (2010). "Nociception". Guide to Research Techniques in Neuroscience. Burlington, MA: Academic Press. pp. 51–2. ISBN 978-0-12-374849-2.

[rd-2] 1 2 3 4 5 6 7 8 9 10 11 12 13 14 Mogil, Jeffrey; Wilson, Sonya; Wan, You (2001). "Assessing Nociception in Murine Subjects". Methods in Pain Research. Frontiers in Neuroscience. Vol. 20012652. doi:10.1201/9781420042566-c2. ISBN 978-0-8493-0035-6.

[3] Koster, R.; Anderson, M.; De Beer, J. (1959). "Acetic acid for analgesic screening". Federation Proceedings. 18: 412–417.

[MateraFlammini2014-4] Matera, Carlo; Flammini, Lisa; Quadri, Marta; Vivo, Valentina; Ballabeni, Vigilio; Holzgrabe, Ulrike; Mohr, Klaus; De Amici, Marco; Barocelli, Elisabetta; Bertoni, Simona; Dallanoce, Clelia (2014). "Bis(ammonio)alkane-type agonists of muscarinic acetylcholine receptors: Synthesis, in vitro functional characterization, and in vivo evaluation of their analgesic activity". European Journal of Medicinal Chemistry. 75: 222–232. doi:10.1016/j.ejmech.2014.01.032. ISSN 0223-5234. PMID 24534538.

[5] Bradman, Matthew J.G.; Ferrini, Francesco; Salio, Chiara; Merighi, Adalberto (November 2015). "Practical mechanical threshold estimation in rodents using von Frey hairs/Semmes–Weinstein monofilaments: Towards a rational method". Journal of Neuroscience Methods. 255: 92–103. doi:10.1016/j.jneumeth.2015.08.010. PMID 26296284. S2CID 206270382.

[cd-6] 1 2 Bannon, Anthony W.; Malmberg, Annika B. (2007). Models of Nociception: Hot-Plate, Tail-Flick, and Formalin Tests in Rodents. Vol. Chapter 8. pp. Unit 8.9. doi:10.1002/0471142301.ns0809s41. ISBN 978-0-471-14230-0. PMID 18428666. S2CID 19332207.{{cite book}}: |journal= ignored (help)

[assay-7] Varnado-Rhodes, Y; Gunther, J; Terman, GW; Chavkin, C (2000). "Mu opioid analgesia and analgesic tolerance in two mouse strains: C57BL/6 and 129/SvJ". Proceedings of the Western Pharmacology Society. 43: 15–7. PMID 11056944.

[8] Lariviere, William R; Wilson, Sonya G; Laughlin, Tinna M; Kokayeff, Anna; West, Erin E; Adhikari, Seetal M; Wan, You; Mogil, Jeffrey S (2002). "Heritability of nociception. III. Genetic relationships among commonly used assays of nociception and hypersensitivity". Pain. 97 (1–2): 75–86. doi:10.1016/S0304-3959(01)00492-4. PMID 12031781. S2CID 17419719.

[9] Caterina, M. J.; Leffler, A; Malmberg, AB; Martin, WJ; Trafton, J; Petersen-Zeitz, KR; Koltzenburg, M; Basbaum, AI; Julius, D (2000). "Impaired Nociception and Pain Sensation in Mice Lacking the Capsaicin Receptor". Science. 288 (5464): 306–13. Bibcode:2000Sci...288..306C. doi:10.1126/science.288.5464.306. PMID 10764638.

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