Double and triple spikes in C-nociceptors in neuropathic pain states: An additional peripheral mechanism of hyperalgesia Jordi Serra a,⇑ , Romà Solà a , Jordi Aleu a , Cristina Quiles a , Xavier Navarro b , Hugh Bostock a,c a Neuroscience Technologies, Barcelona, Spain b Grup de Neuroplasticitat i Regeneració, Departament de Biologia Cellular, Fisiologia i Inmunologia, Institut de Neurociències, Universitat Autònoma de Barcelona, Barcelona, Spain c Institute of Neurology, University College London, London, UK article info Article history: Received 25 January 2010 Received in revised form 20 September 2010 Accepted 27 October 2010 Keywords: Microneurography Hyperalgesia Allodynia Animal models Pain Action potential abstract It was previously reported that in 5 patients with small-fiber neuropathy, neuropathic pain, and hyper- algesia, application of a single, brief electrical stimulus to the skin could give rise to 2 afferent impulses in a C-nociceptor fiber. These double spikes, which are attributed to unidirectional conduction failure at branch points in the terminal arborisation, provide a possible mechanism for hyperalgesia. We here report that similar multiple spikes are regularly observed in 3 rat models of neuropathic pain: nerve crush, nerve suture, and chronic constriction injury. The proportion of nociceptor fibers exhibiting multi- ple spikes was similar (10.1–18.5%) in the 3 models, and significantly greater than the proportion in con- trol (unoperated) animals (1.2%). As in the human patients, multiple spikes in the rat models were often provoked by increasing the stimulation rate from 0.25 to 2 Hz, but provocation by warming was less con- sistent. Multiple spiking was also directly dependent on stimulus intensity, consistent with a mechanism that depends on activation of multiple branches. Whereas only double spikes had previously been described in patients, in these more extensive recordings from rats we found that triple spikes could also be observed after a single electrical stimulus. The results strengthen the suggestion that multiple spiking, because of impaired conduction in the terminal branches of nociceptors, may contribute to hyperalgesia in patients with neuropathic pain. Ó 2010 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved. 1. Introduction Neuropathic pain patients frequently exhibit increased evoked pain in response to cutaneous stimulation, in the form of hyperal- gesia, which is an increased pain to a normally painful stimulus, or allodynia, defined as pain to a normally nonpainful stimulus [7,11]. This increased responsiveness is due to a ‘‘gain’’ in the abnormally functioning somatosensory pathways. Hypothetically, this gain may occur at different levels along the sensory pathways, from the periphery to the central nervous system. Central sensitization of dorsal horn neurons has been proposed as an important cause for this increased responsiveness to afferent inputs [15]. However, it is known that this abnormal gain can also occur because of mechanisms operating at a peripheral level. For example, lowering of the peripheral nociceptor receptor thresholds, a phenomenon named peripheral sensitization has been shown to underlie the pri- mary hyperalgesia that occurs at a site of cutaneous injury [14,17] and also the increased skin sensitivity found in some neuropathic pain patients [6,8]. Recently, a possible new mechanism for peripheral amplifica- tion of sensory inputs has been reported. Under normal conditions, single electrical pulses delivered to the terminal branches of cuta- neous C-nociceptors induce single spikes conducted to the central nervous system. This occurs despite the fact that single electrical pulses may initiate action potentials at several different sites in the terminal branches of a nociceptor. Only the ‘‘leading’’ action potential, generated closest to the main parent axon, is conducted centrally, and the rest are cancelled by collision from antidromi- cally conducted action potentials (see Section 4). However, Weidner et al. [26] reported that this is not always the case in a small proportion of healthy subjects (3%). They found C-nociceptor units in normal subjects which sometimes conducted 2 impulses after a single brief stimulus, and when they did so, the profile of activity-dependent slowing was appropriate for 2 impulses rather than 1. They argued that these double spikes orig- inated from different terminal branches and required a unidirec- tional conduction block to prevent the faster action potential from invading and resetting the slower-conducting terminal branches, constituting a possible peripheral mechanism for pain 0304-3959/$36.00 Ó 2010 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.pain.2010.10.039 ⇑ Corresponding author. Address: Neuroscience Technologies, Barcelona Science Park, c. Baldiri Reixac 15-21, 08028 Barcelona, Spain. Tel./fax: +34 93 402 0164. E-mail address: jserra@nsc-tec.com (J. Serra). PAIN Ò 152 (2011) 343–353 www.elsevier.com/locate/pain