Gabapentin has a prominent antihyperalgesic action after either systemic or spinal (intrathecal: IT) delivery.1 – 5 Previous work emphasized that the molecule binds with high affinity to the α2δ1 extracellular auxiliary subunit of voltage-sensitive calcium channels (VSCCs), particularly the N- and L-, but not T-, types.6,7 Point mutation studies of the α2δ1 protein showed that such changes had no effect upon calcium channel function, but completely prevented the binding and actions of gabapentin.8 Although the analgesic efficacy of gabapentin in various facilitated pain states is well known, its mechanism of action remains uncertain. Given the structural link between α2δ1 and the pore-forming elements of the VSCCs and the presence of α2δ1 in dorsal root ganglion (DRG),9 a prevalent hypothesis is that gabapentin regulates afferent terminal transmitter release through regulation of calcium channel function. However, this hypothesis is complex. Thus, although an acute regulation by gabapentin of DRG calcium currents has been reported,10 – 12 gabapentin had only modest effects upon the frequency and amplitude of excitatory postsynaptic currents in superficial dorsal horn neurons.13 That work suggests that ongoing presynaptic neurotransmitter release is not regulated by this drug. Direct measurement of release in spinal slices has shown that gabapentin can diminish release of glutamate and several peptides,14,15 though with the caveat that the effects appear limited to conditions associated with prior sensitization or lengthy drug exposures.16,17 Other work has emphasized that gabapentin's actions may be dependent upon the up-regulation of α2δ1 as occurs after nerve injury9 or may alter calcium channel trafficking.18 Such hypotheses imply the likelihood that the antihyperalgesic actions of gabapentin are limited to events that may require exposures of hours to days. Yet it is evident from the acute antihyperalgesic effects of gabapentin in vivo that other acute mechanisms must be in play. The present experiments aimed to determine the effects of gabapentin in vivo on the spinal release of substance P evoked by intraplantar formalin injection. Because dorsal horn substance P is primarily contained in small TRPV1 (+) (transient receptor potential vanilloid 1 receptor) primary afferents, changes in its release reflect an effect upon the releasing function of that small afferent terminal, in contrast to a transmitter such as glutamate, which may arise from many neurons and even glia. In the present work, substance P release was examined in vivo by assessing the internalization of the neurokinin/receptor (NK1r) in the superficial dorsal horn produced by unilateral intraplantar formalin injection. NK1rs are densely located on lamina I projection neurons, but are largely absent from lamina II neurons.19,20 Finally, we sought to compare the effects of gabapentin given IT and intraperitoneally (IP) at equianalgesic doses. Previous work with morphine interestingly demonstrated that internalization was blocked at analgesic doses after IT delivery, but supra-analgesic doses after systemic delivery.21 As intraplantar formalin injection evokes a well-defined biphasic flinching response, the in vivo activity of gabapentin on release and pain behavior can be compared.
Male Holtzman Sprague–Dawley rats (250 to 300 g; Harlan, Indianapolis, IN) were individually housed and maintained on a 12-hour light/dark cycle with testing occurring only during the light cycle. Food and water were available ad libitum. Animal care was in accordance with the Guide for the Care and Use of Laboratory Animals (National Institutes of Health publication 85 to 23, Bethesda, MD) and as approved by the institutional Animal Care and Use Committee of the University of California, San Diego.
Intrathecal Catheter Implantation
Rats were implanted with a single IT catheter for drug delivery.22,23 In brief, rats anesthetized with isoflurane in an air/oxygen mixture (1:1) were implanted with a polyethylene (outside diameter 0.36 mm) catheter passed through the cisternal membrane into the IT space to the level of the L2 to 3 spinal segments (approximately 8.5 cm) and externalized on top of the head. Animals were given 5 mL of lactated Ringer's solution subcutaneously, and they recovered under a heat lamp. Rats without motor deficit were allowed to recover for 5 to 7 days before study.
Formalin-Induced Paw-Flinching Behavior
To assess formalin-evoked flinching, we placed a metal band around the left hindpaw of the animal, and we allowed the rat to acclimate 30 minutes before experimental manipulation. At time 0, formalin (5%, 50 μL) was delivered into the left hindpaw. Immediately after the formalin injection, rats were placed into the test chamber and nociceptive behavior (flinching and shaking of the injected paw) was quantified by an automatic flinch counting device (UARDG, Department of Anesthesiology, University of California, San Diego).24 Flinches were counted in 1-minute intervals for 60 minutes. The data are expressed as total number of flinches observed during phase 1 (0 to 10 minutes) and phase 2 (11 to 60 minutes).
Drugs and Delivery
Gabapentin (Sigma Chemical, St. Louis, MO) was dissolved in 0.9% saline to be delivered in a volume of 10 μL for IT delivery or to be delivered IP in a volume of 1.5 mL/kg. Drugs were administered IT 10 minutes or IP 15 minutes before the intraplantar delivery of formalin (5%, 50 μL).
Effects of Intrathecal Gabapentin on NK1r Internalization Induced by Exogenous Substance P
To exclude the possibility that gabapentin directly blocked the NK1r internalization mechanism, we administered IT saline or gabapentin to rats before IT substance P (30 nmol). Thirty minutes later, rats were killed and fixed. Spinal NK1r immunoreactive neurons, with or without NK1r internalization, were counted.
Tissue Preparation and Immunocytochemistry
For tissue harvest (10 minutes after intraplantar formalin), rats were deeply anesthetized with isoflurane and transcardially perfused with 0.9% NaCl, followed by 4% paraformaldehyde in 0.1 M sodium phosphate buffer (PBS), pH 7.4. The lumbar cord was removed and postfixed overnight. After cryoprotection in 30% sucrose, coronal sections were taken using a sliding microtome (30 μm). To assess NK1r expression in the spinal cord dorsal horn, we incubated sections with a rabbit anti-NK1r polyclonal antibody (1:3000 in 0.01 M PBS containing 10% normal goat serum and 10% Triton X-100 (Advanced Targeting Systems, San Diego, CA) overnight at room temperature. After a rinse in PBS, sections were incubated for 120 minutes at room temperature in a goat antirabbit secondary antibody conjugated with Alexa Fluor 488 (1:1000 in same buffer; Invitrogen, Eugene, OR). All sections were rinsed, mounted on glass slides, and coverslipped with ProLong mounting medium (Fisher Scientific, Pittsburgh, PA).
Quantification of NK1r Internalization
NK1r internalization was counted using an Olympus BX-51 fluorescence microscope (Olympus Optical, Tokyo, Japan), and a 60× oil-immersion objective lens and internalization was assessed according to the standard of previous reports.19,25 The filter set was Omega Optical XF100 to 2 Green Bandpass Filter. Neuronal profiles that had 10 or more endosomes were considered to have internalized NK1rs. The total number of NK1r immunoreactive neurons in lamina I/II, with and without NK1r internalization, was counted and taken to calculate the fraction of cells showing internalization. Counting was done without knowledge of treatments. Mean counts from 2 to 5 sections per segment were used as representative counts for a given animal.26 Images were taken using Magna FIRE SP and processed by Adobe Photoshop CS4. Nomenclature for drugs and receptors conforms with the guide to receptors and channels of the British Journal of Pharmacology.27
Changes in paw-flinching behavior were analyzed using 1-way analysis of variance (ANOVA) for phases 1 and 2 with Tukey post hoc tests performed for pairwise comparisons. Analysis of NK1r internalization data consisted of 1-way or 2-way ANOVAs with Tukey post hoc analysis. Significance was defined as P < 0.05.
Effects of Intrathecal and Intraperitoneal Gabapentin on Formalin Evoked NK1r Internalization
NK1r immunoreactivity was typically observed outlining the cytosolic membrane in many superficial dorsal horn neurons (Fig. 1B). Unilateral intraplantar injection of formalin (5%, 50 μL) produced a robust NK1r internalization in ipsilateral superficial dorsal horn (Figs. 1A, 2C) but not contrallateral dorsal horn (ipsilateral L4: 26 ± 5%, L5: 62 ± 5%, L6: 62 ± 8%; contralateral L4: 12 ± 4%, L5: 5 ± 2%, L6: 14 ± 6%, P < 0.05). IT gabapentin (200 μg but not 100 μg) showed a prominent reduction in the ipsilateral L5 and L6 increase in formalin-evoked internalization (P < 0.05) (Fig. 2C). IP gabapentin (200 mg/kg, but not 100 mg/kg) significantly reduced the formalin-induced NK1r internalization at L5 and L6 levels of the superficial lumbar spinal cord (P < 0.05) (Fig. 3C).
Effects of Intrathecal Gabapentin on NK1r Internalization Induced by Exogenous Substance P
IT substance P (30 nmol) produced widespread NK1r internalization at L4 to 6 levels of spinal cord lamina I in comparison with IT saline (P < 0.05) (Fig. 4, A–C). IT gabapentin (200 μg), a dose that completely blocked formalin-induced NK1r internalization, did not affect exogenous substance P–induced NK1r internalization (Fig. 4, B and D).
Effects of Intrathecal and Intraperitoneal Gabapentin on Formalin Evoked Flinching Behavior
Intraplantar formalin resulted in a robust biphasic incidence of flinching over a 60-minute period. Pretreatment with IT gabapentin (100 and 200 μg) resulted in a dose-dependent suppression of phase 2 flinching with no effect on phase 1, in comparison with vehicle-treated control (Fig. 2, A and B). Similarly, after IP delivery, gabapentin (100 and 200 mg/kg) diminished phase 2, but not phase 1, in comparison with vehicle control (Fig. 3, A and B). Importantly, IT gabapentin at the highest dose did not produce adverse effects on behavior or motor function, in comparison with vehicle control (Table 1). IP 100 and 200 mg/kg gabapentin were observed to be associated with reduced spontaneous activity in some rats. No other motor effects were noted.
As reviewed, binding to the α2δ1subunit associated with VSCCs is likely essential for the spinal antihyperalgesic actions of gabapentin and its analogues.28 The association of α2δ1subunits with VSCCs in DRGs, along with the likelihood that one or more of these VSCCs may be instrumental in mediating depolarization-evoked transmitter release, raises the possibility that gabapentin may regulate release from primary afferents.29
Afferent Substance P Release
The present results indeed indicate that gabapentin given either IT or IP exerts a potent, short latencied effect in vivo upon small afferent-evoked substance P release. Previous work showed that IT gabapentin reduced formalin-induced release of glutamate in spinal dorsal horn30 and systemic gabapentin reduced spinal glutamate release after a visceral irritant.31 However, unlike the present work with substance P, the effects of gabapentin on spinal glutamate release cannot necessarily be ascribed to primary afferents, given the ubiquitous nature of spinal glutamate distribution. The present effects of gabapentin on substance P release are in accordance with recent work in which we showed that substance P release in spinal dorsal horn can be attenuated in vivo by IT N-type, but not T- or L-type, calcium channel blockers.32
The use of NK1r internalization as an index of primary afferent substance P release has been validated by several criteria and reviewed previously.21,33 An important caveat is the concern that the agent has a direct effect upon the postsynaptic internalization process. We showed that internalization produced by exogenous IT substance P was, however, not altered by IT gabapentin, indicating that the suppressant effects on evoked release was not due to a direct effect on the internalization process.
Facilitated Pain States
The present work confirmed a potent antinociceptive effect of gabapentin given IT or IP upon phase 2, but not phase 1, of the formalin-evoked flinching, a finding consistent with those previously reported.1 – 5 Consistent with a spinal action of gabapentin, the effective total dose was 200 μg IT versus 60 mg IP (e.g., total dose for 200 mg/kg in a 300-g rat).
Correlation Between Effects on Substance P Release and Antinociception
The functional relevance of the IT/IP gabapentin effects on release is suggested by the parallel between behaviorally relevant doses and those required to reduce release. We note, however, that after both IT and IP delivery, phase 2 flinching was reduced at doses lower than those required to produce reduction in NK1r internalization. Whether this represents a component of gabapentin's action on the flinching model, which is not mediated by effects on peptidergic C-fibers, an effect upon other spinal systems, or even nonspinal systems34 is not known.
Effect of Gabapentin on Acute Facilitation
One feature of gabapentin's actions is its effects upon facilitated pain states, which may be initiated within minutes of stimulation (phase 2 flinching and tissue injury-evoked hyperalgesia) but not upon acute nociception (phase 1 flinching, thermal thresholds).1 – 5,35 An example of the acuity of this effect is the observation that although IT or IP gabapentin or pregabalin (S(+)-3-isobutyl-γ aminobutyric acid) has no effect upon acute thermal escape latencies, they reverse the thermal hyperalgesia evoked in seconds to minutes by IT substance P, an effect reflecting a postsynaptic effect mediated by substance P on the NK1r. The specificity of this effect is emphasized by the observation that an isomer of pregabalin (R(-)-3-isobutyl-γ aminobutyric acid) with lower α2δ1 binding affinity was less active in vivo.36 This differential effect upon acute versus facilitated states distinguishes the actions of gabapentin from those of other drugs that block small afferent release, such as opiates.37 The observation that gabapentin did not block NK1r internalization evoked by IT substance P, and yet blocked the hyperalgesia evoked by IT substance P, raises the interesting likelihood that gabapentin has several sites of spinal action, one of which is presynaptic on the afferent (to block release of substance P and likely other afferent transmitters such as glutamate) and one of which appears not to be on the primary afferent.13 Where the nonafferent action is we do not know, but the lack of effect upon internalization evoked by IT substance P emphasizes that the gabapentin effect was not due to an artifact of an action on the internalization process.
Several mechanisms for this acute action on facilitated states present themselves. (i) The effects upon presynaptic release initiated by persistent small afferent input, as generated during the first phase,38 may represent distinct processes in the presynaptic terminal leading to release. Protein kinase C and adenylyl cyclase activation exert a potent facilitatory effect upon glutamate release in trigeminal slices, and this facilitation (but not acute release) is blocked by gabapentin, perhaps through reduced phosphorylation of associated VSCCs.14 (ii) α2δ1subunits are distributed in a variety of DRG cells.18 Accordingly, there are likely effects upon terminals that are not peptidergic. (iii) In dorsal horn, α2δ1 may be associated with nonafferent terminals. As has been noted, gabapentin blocks hyperalgesia initiated by spinal NK1 activation.36 In this vein, gabapentin blocks substance P–evoked NF-κB activation in cell systems by blockade of IkB-α degradation, thereby preventing translocation of p65 to the nucleus.39
Mechanisms of Actions of Gabapentins
As is discussed above, the membrane level, the role of the binding α2δ1 site in directly regulating calcium channel function, has been controversial. The present studies, however, do emphasize the presynaptic effect upon small afferent terminal-releasing properties. Other mechanisms that may be associated with the actions of gabapentin include the trafficking of intracellular VSCC, prevented by gabapentin,18 and spouting after nerve injury. Thus, α2δ1binds the thrombospondin family of extracellular matrix proteins, which play a role in intracellular trafficking and synaptogenesis.40,41 Importantly, these proteins are secreted by glia.40 Previous work has indicated that even acute tissue injury or IT substance P can initiate glial activation as measured by, for example, phosphorylation of glial P38 mitogen-activated protein kinase.42
As is outlined above, however, an important element in the present study is that it draws attention to the acuity (seconds to minutes) of the biological effects of gabapentin, including suppression of the release of at least 1 small afferent transmitter and the acute changes in facilitated spinal states. Thus, there is little doubt that up-regulation of the α2δ1 subunits occurs after nerve injury43 and that chronic gabapentin exposure may lead to a down-regulation of the associated VSCCs16 or other intracellular changes17 that alter VSCCs insertion into the cell membrane. The acute manifestations observed in vivo clearly represent an effect upon a constitutively expressed and functional spinal system that does not require additional changes in protein expression or channel mobilization or extended drug exposure.9 This profile of action thus forces us to consider the likelihood that additional mechanisms may be called into play that define the regulation exerted at the spinal level in vivo by the α2δ1 binding exhibited by gabapentin and its homologues.
In conclusion, the present work emphasizes that IT and systemic administration of gabapentin acutely regulate spinal release of substance P from small primary afferents at doses that are associated with its antihyperalgesic action. These results, along with previous electrophysiological13 and behavioral36 work consistent with a postsynaptic action, suggest that a number of mechanisms may reflect the acute actions of gabapentin and indirectly the role of the spinal α2δ1 subunit to which it binds.
Name: Toshifumi Takasusuki, MD, PhD.
Contribution: Conduct of study and data analysis.
Name: Tony L. Yaksh, PhD.
Contribution: Study design and manuscript preparation.
We would like to thank Arbi Nazarian, PhD, for his assistance in setting up the internalization protocol.
1. Field MJ, Oles RJ, Lewis AS, McCleary S, Hughes J, Singh L. Gabapentin (neurontin) and S-(+)-3-isobutylgaba represent a novel class of selective antihyperalgesic agents. Br J Pharmacol 1997;121:1513–22
2. Hwang JH, Yaksh TL. Effect of subarachnoid gabapentin on tactile-evoked allodynia in a surgically induced neuropathic pain model in the rat. Reg Anesth 1997;22:249–56
3. Yoon MH, Yaksh TL. Evaluation of interaction between gabapentin and ibuprofen on the formalin test in rats. Anesthesiology 1999;91:1006–13
4. Yoon MH, Yaksh TL. The effect of intrathecal gabapentin on pain behavior and hemodynamics on the formalin test in the rat. Anesth Analg 1999;89:434–9
5. Ortega-Varela LF, Herrera JE, Caram-Salas NL, Rocha-González HI, Granados-Soto V. Isobolographic analyses of the gabapentin–metamizol combination after local peripheral, intrathecal and oral administration in the rat. Pharmacology 2007;79:214–22
6. Stefani A, Spadoni F, Bernardi G. Gabapentin inhibits calcium currents in isolated rat brain neurons. Neuropharmacology 1998;37:83–91
7. Davies A, Hendrich J, Van Minh AT, Wratten J, Douglas L, Dolphin AC. Functional biology of the alpha (2) delta subunits of voltage-gated calcium channels. Trends Pharmacol Sci 2007;28:220–8
8. Field MJ, Cox PJ, Stott E, Melrose H, Offord J, Su TZ, Bramwell S, Corradini L, Engl S, Winks J, Kinloch RA, Hendrich J, Dolphin AC, Webb T, Williams D. Identification of the alpha2-delta-1 subunit of voltage-dependent calcium channels as a molecular target for pain mediating the analgesic actions of pregabalin. Proc Natl Acad Sci USA 2006;103:17, 537–42
9. Luo ZD, Chaplan SR, Higuera ES, Sorkin LS, Stauderman KA, Williams ME, Yaksh TL. Upregulation of dorsal root ganglion (alpha) 2 (delta) calcium channel subunit and its correlation with allodynia in spinal nerve–injured rats. J Neurosci 2001;21:1868–75
10. Alden KJ, García J. Differential effect of gabapentin on neuronal and muscle calcium currents. J Pharmacol Exp Ther 2001;297:727–35
11. Sarantopoulos C, McCallum B, Kwok WM, Hogan Q. Gabapentin decreases membrane calcium currents in injured as well as in control mammalian primary afferent neurons. Reg Anesth Pain Med 2002;27:47–57
12. Li CY, Zhang XL, Matthews EA, Li KW, Kurwa A, Boroujerdi A, Gross J, Gold MS, Dickenson AH, Feng G, Luo ZD. Calcium channel alpha2delta1 subunit mediates spinal hyperexcitability in pain modulation. Pain 2006;125:20–34
13. Moore KA, Baba H, Woolf CJ. Gabapentin– actions on adult superficial dorsal horn neurons. Neuropharmacology 2002;43:1077–81
14. Maneuf YP, Hughes J, McKnight AT. Gabapentin inhibits the substance P-facilitated K(+)-evoked release of [(3)H]glutamate from rat caudial trigeminal nucleus slices. Pain 2001;93:191–6
15. Fehrenbacher JC, Taylor CP, Vasko MR. Pregabalin and gabapentin reduce release of substance P and CGRP from rat spinal tissues only after inflammation or activation of protein kinase C. Pain 2003;105:133–41
16. Vega-Hernández A, Felix R. Down-regulation of N-type voltage-activated Ca2+ channels by gabapentin. Cell Mol Neurobiol 2002;22:185–90
17. Heblich F, Tran Van Minh A, Hendrich J, Watschinger K, Dolphin AC. Time course and specificity of the pharmacological disruption of the trafficking of voltage-gated calcium channels by gabapentin. Channels (Austin). 2008;2:4–9
18. Bauer CS, Nieto-Rostro M, Rahman W, Tran Van Minh A, Ferron L, Douglas L, Kadurin I, Sri Ranjan Y, Fernandez-Alacid L, Millar NS, Dickenson AH, Lujan R, Dolphin AC. The increased trafficking of the calcium channel subunit alpha2delta-1 to presynaptic terminals in neuropathic pain is inhibited by the alpha2delta ligand pregabalin. J Neurosci 2009;29:4076–88
19. Mantyh PW, Allen CJ, Ghilardi JR, Rogers SD, Mantyh CR, Liu H, Basbaum AI, Vigna SR, Maggio JE. Rapid endocytosis of a G protein-coupled receptor: substance P evoked internalization of its receptor in the rat striatum in vivo. Proc Natl Acad Sci USA 1995;92:2622–6
20. Willis WD Jr. The somatosensory system, with emphasis on structures important for pain. Brain Res Rev 2007;55:297–313
21. Kondo I, Marvizon JC, Song B, Salgado F, Codeluppi S, Hua XY, Yaksh TL. Inhibition by spinal mu- and delta-opioid agonists of afferent-evoked substance P release. J Neurosci 2005;25:3651–60
22. Yaksh TL, Rudy TA. Chronic catheterization of the spinal subarachnoid space. Physiol Behav 1976;17:1031–6
23. Malkmus SA, Yaksh TL. Intrathecal catheterization and drug delivery in the rat. Methods Mol Med 2004;99:109–21
24. Yaksh TL, Ozaki G, McCumber D, Rathbun M, Svensson C, Malkmus S, Yaksh MC. An automated flinch detecting system for use in the formalin nociceptive bioassay. J Appl Physiol 2001;90:2386–402
25. Abbadie C, Trafton J, Liu H, Mantyh PW, Basbaum AI. Inflammation increases the distribution of dorsal horn neurons that internalize the neurokinin-1 receptor in response to noxious and non-noxious stimulation. J Neurosci 1997;17:8049–60
26. Nazarian A, Christianson CA, Hua XY, Yaksh TL. Dexmedetomidine and ST-91 analgesia in the formalin model is mediated by alpha2A-adrenoceptors: a mechanism of action distinct from morphine. Br J Pharmacol 2008;155:1117–26
27. Alexander SP, Mathie A, Peters JA. Guide to Receptors and Channels (GRAC), 3rd ed. Br J Pharmacol 2008;153:S1–09
28. Maneuf YP, Gonzalez MI, Sutton KS, Chung FZ, Pinnock RD, Lee K. Cellular and molecular action of the putative GABA-mimetic, gabapentin. Cell Mol Life Sci 2003;60:742–50
29. Boroujerdi A, Kim HK, Lyu YS, Kim DS, Figueroa KW, Chung JM, Luo ZD. Injury discharges regulate calcium channel α2δ1 subunit upregulation in the dorsal horn that contributes to initiation of neuropathic pain. Pain 2009;139:358–66
30. Coderre TJ, Kumar N, Lefebvre CD, Yu JS. Evidence that gabapentin reduces neuropathic pain by inhibiting the spinal release of glutamate. J Neurochem 2005;94:1131–9
31. Feng Y, Cui M, Willis WD. Gabapentin markedly reduces acetic acid-induced visceral nociception. Anesthesiology 2003;98:729–33
32. Takasusuki T, Xu Q, Yaksh TL. Regulation of spinal substance Prelease by intrathecal calcium channel blockade and gabapentin. 13th
World Congress on Pain 2010 10-A-1669
33. Marvizón JC, Wang X, Matsuka Y, Neubert JK, Spigelman I. Relationship between capsaicin-evoked substance P release and neurokinin 1 receptor internalization in the rat spinal cord. Neuroscience 2003;118:535–45
34. Tanabe M, Takasu K, Takeuchi Y, Ono H. Pain relief by gabapentin and pregabalin via supraspinal mechanisms after peripheral nerve injury. J Neurosci Res 2008;86:3258–64
35. Tuchman M, Barrett JA, Donevan S, Hedberg TG, Taylor CP. Central sensitization and Ca(V)alpha(2)delta ligands in chronic pain syndromes: pathologic processes and pharmacologic effect. J Pain 2010;11:1241–9
36. Partridge BJ, Chaplan SR, Sakamoto E, Yaksh TL. Characterization of the effects of gabapentin and 3-isobutyl-gamma-aminobutyric acid on substance P-induced thermal hyperalgesia. Anesthesiology 1998;88:196–205
37. Yaksh TL. Spinal systems and pain processing: development of novel analgesic drugs with mechanistically defined models. Trends Pharmacol Sci 1999;20:329–37
38. Puig S, Sorkin LS. Formalin-evoked activity in identified primary afferent fibers: systemic lidocaine suppresses phase-2 activity. Pain 1996;64:345–55
39. Park S, Ahn ES, Han DW, Lee JH, Min KT, Kim H, Hong YW. Pregabalin and gabapentin inhibit substance P-induced NF-kappaB activation in neuroblastoma and glioma cells. J Cell Biochem 2008;105:414–23
40. Eroglu C, Allen NJ, Susman MW, O'Rourke NA, Park CY, Ozkan E, Chakraborty C, Mulinyawe SB, Annis DS, Huberman AD, Green EM, Lawler J, Dolmetsch R, Garcia KC, Smith SJ, Luo ZD, Rosenthal A, Mosher DF, Barres BA. Gabapentin receptor alpha2delta-1 is a neuronal thrombospondin receptor responsible for excitatory CNS synaptogenesis. Cell 2009;139:380–92
41. Kurshan PT, Oztan A, Schwarz TL. Presynaptic alpha2delta-3 is required for synaptic morphogenesis independent of its Ca2+-channel functions. Nat Neurosci 2009;12:1415–23
42. Svensson CI, Marsala M, Westerlund A, Calcutt NA, Campana WM, Freshwater JD, Catalano R, Feng Y, Protter AA, Scott B, Yaksh TL. Activation of p38 mitogen-activated protein kinase in spinal microglia is a critical link in inflammation-induced spinal pain processing. J Neurochem 2003;86:1534–44
43. Luo ZD, Calcutt NA, Higuera ES, Valder CR, Song YH, Svensson CI, Myers RR. Injury type-specific calcium channel alpha 2 delta-1 subunit up-regulation in rat neuropathic pain models correlates with antiallodynic effects of gabapentin. J Pharmacol Exp Ther 2002;303:1199–205