Recent studies have suggested a relationship between neuropathic pain and the endogenous pain inhibitory system.1,2 Patients with established chronic pain tend to present attenuated endogenous analgesia compared with pain-free individuals.3 Endogenous analgesia in humans can be evaluated by measuring conditioned pain modulation.4 In rats, conditioned pain modulation typically is assessed by the use of noxious stimulus–induced analgesia (NSIA).5 The noxious stimulus in NSIA is subcutaneous injection of capsaicin, and the resulting endogenous analgesia is heterosegmental, in that antinociception can be observed at sites remote from the site of stimulation.6 For example, capsaicin injection into the forepaw elevates hindpaw withdrawal thresholds in response to mechanical stimulation.5 NSIA can produce analgesia equivalent to that of a high dose of morphine (10 mg/kg) and lasts approximately 1 hour.6,7
Recent studies have demonstrated that the descending noradrenergic system is involved in NSIA; capsaicin injection into the forepaw was shown to increase the noradrenaline level in the lumbar spinal cord,5,8 and NSIA was blocked partially by intrathecal injection of an α2-adrenergic antagonist and by ablation of the descending noradrenergic innervation of the spinal cord.5 The brain-spinal descending noradrenergic system suppresses nociceptive signals from primary afferent neurons to the spinal dorsal horn, and the inhibitory system also plays an important role in neuropathic pain states.9 Previous studies also demonstrated that the tone of the descending noradrenergic system changes over time after nerve injury.10–12 Therefore, we hypothesized that NSIA is modulated by the plasticity of descending noradrenergic systems after nerve injury. Thus, the first purpose of this study was to examine the change of NSIA over time in a rat model of neuropathic pain induced by spinal nerve ligation (SNL).
Antidepressants, such as tricyclic antidepressants and pregabalin, are used as first-line drugs for treatment of neuropathic pain,13 and previous studies demonstrated that the noradrenergic system is strongly involved in the analgesic effect of gabapentinoids14–16 and amitriptyline17 after peripheral nerve injury. Therefore, we hypothesized that amitriptyline and pregabalin also would modulate the effect of NSIA in SNL rats, and the second purpose of this study was to examine the change in NSIA and noradrenaline release in the spinal cord after 5 daily intraperitoneal injections of amitriptyline or pregabalin in rats with SNL.
Anatomical relationships and the time course of the experiments are diagrammed in Figure 1.
This experiment was approved by the Animal Care and Use Committee of Gunma University Graduate School of Medicine (Maebashi, Japan). Male Sprague-Dawley rats (250 g; SLC, Shizuoka, Japan) were used in all experiments. Animals were housed under a 12-hour light-dark cycle, with food and water provided ad libitum.
We used the L5 SNL model as an experimental model of neuropathic pain as described by Kim and Chung.18 In brief, the animals were anesthetized with inhaled isoflurane in oxygen, and the right L5 spinal nerve was tightly ligated with 6-0 silk and cut just distal to the ligature.
Withdrawal thresholds were measured by testing of the withdrawal response to a noxious pressure stimulus (Randall-Selitto test) using an analgesimeter (37215; Ugo Basile, Comerio, Italy), as previously described.19 A cutoff of 250 g was used to prevent potential tissue injury. All animals were trained for 3 days with the device before baseline values were recorded. Experiments with normal and SNL rats were performed separately. The experimenter was unaware of the drug and dose.
NSIA was elicited by subcutaneous injection of capsaicin (250 μg in a volume of 50 μL) administered to the left forepaw during brief general anesthesia induced by isoflurane (2%) inhalation. The degree of analgesia in the left hind paw was assessed by withdrawal thresholds as described previously. At each time point, withdrawal thresholds were assessed twice on the plantar aspect of left hind paw, and the results were averaged. NSIA was assessed in normal rats and SNL rats. SNL rats were evaluated at 1, 4, 5, and 6 weeks after SNL surgery. Capsaicin was initially dissolved in Tween 80 (50%) and ethanol (50%) to a concentration of 50 μg/μL and then diluted with saline to a concentration of 5 μg/μL; the subcutaneous capsaicin injection volume was 50 μL (250 μg) in all experiments.
Effect of Amitriptyline and Pregabalin on NSIA
Rats received 5 daily intraperitoneal injections of amitriptyline (10 mg/kg/d) or pregabalin (10 mg/kg/d) from 5 weeks (35 days) after SNL surgery. The bilateral paw withdrawal threshold was measured every day 30 minutes before drug injection to evaluate the chronic effects of the drugs. At 6 weeks (42 days) after SNL surgery, NSIA was assessed as described previously. Amitriptyline and pregabalin were dissolved in saline and intraperitoneally injected at 10 mg/kg for 5 consecutive days from 5 weeks after SNL surgery. In the control group, saline (1 mL) was injected rather than amitriptyline or pregabalin under the same schedule. Amitriptyline was purchased from LKT Laboratories, Inc (St. Paul, MN), and pregabalin was donated by Pfizer Inc (Groton, CT).
Microdialysis studies were performed to measure noradrenaline levels after left forepaw capsaicin injection in the left spinal dorsal horn as described previously.20 Anesthesia was induced using 5% isoflurane and maintained with 1.5% isoflurane in 100% oxygen through a nose cone. The left femoral vein was cannulated for saline infusion (1 mL/h), rectal temperature was maintained at 37 to 38°C, and the Th12 to L1 level of the spinal cord was exposed. The microdialysis probe (outer diameter = 0.22 mm, inner diameter = 0.20 mm, length = 1 mm; A-I-8-01; Eicom Co., Kyoto, Japan) was inserted from just lateral to the dorsal root and advanced at a 20° angle to a depth of 1 mm and perfused with Ringer’s solution at a constant flow rate (1 μL/min).
After 120 minutes of constant perfusion, consecutive samples were collected to determine the basal noradrenaline concentrations in the dialysate. Capsaicin (250 μg) or vehicle (50 μL) was injected subcutaneously to the left forepaw, and 15-minute perfusate fractions were collected into an autoinjector (EAS-20; Eicom Co.). Samples (15 μL) were automatically injected and analyzed for noradrenaline concentration using high-performance liquid chromatography with electrochemical detection by an HTEC-500 analyzing system (Eicom Co.). The sample was separated on the column (2.0 mm × 200 mm, EICOMPAC CAX; Eicom Co.) using a mobile phase consisting of 0.1 M ammonium acetate buffer (pH 6.0) and methanol (7:3 v/v) containing 0.05 M sodium sulfonate and 50 mg/l EDTA-2Na.
We performed a power analysis for our primary outcome (mechanical withdrawal threshold in NSIA) to determine the appropriate sample size, with the assumptions of mean difference in withdrawal threshold of 50 g and SD in each group of 30 g according to a previous study.5 We found that 6 rats in each group would result in detection of significant differences, with 80% power at a significant level of α = 0.05. The effects of the capsaicin injection or the drug treatments on withdrawal thresholds in the behavioral studies and on the spinal cord noradrenaline level in the microdialysis studies were analyzed using 2-way repeated-measures analysis of variance (ANOVA), followed by Student t test with Bonferroni correction. The residuals of each of the 8 ANOVA models followed normal distributions (for all 8 models: P > 0.207 by Shapiro-Wilk test) and maintained equality of variance (for all 8 models: P > 0.082 by Levene test). The data were presented as the mean ± SD. P value <0.01 was considered statistically significant. The statistical analysis was conducted using SigmaPlot 12 (Systat Software Inc, San Jose, CA).
NSIA in Normal Rats
Subcutaneous injection of capsaicin into the left forepaw resulted in an increased withdrawal threshold to mechanical pressure in the ipsilateral hind paw in noninjured rats, with a peak effect at 30 minutes after capsaicin injection and an effect duration of approximately 75 minutes (Figure 2). Two-way repeated-measures ANOVA showed a significant difference between vehicle and capsaicin injection (F1,30 = 252.8; P < 0.001).
NSIA in SNL Rats
Compared with noninjured rats, withdrawal thresholds to mechanical pressure in the left hind paw of SNL rats subcutaneously injected with capsaicin in the ipsilateral forepaw were significantly decreased at 1, 4, 5, and 6 weeks after right SNL (Figure 3; F4, 84 = 64.772; P < 0.001 by 2-way repeated-measures ANOVA). NSIA at 5 and 6 weeks after SNL was significantly decreased compared with that at other time points (P < 0.001 by Student t test with Bonferroni correction).
Spinal Noradrenaline Release in Noninjured Rats and in Rats 6 Weeks After SNL Surgery
The concentration of noradrenaline measured by microdialysis was increased significantly in noninjured rats subcutaneously injected with capsaicin compared with those injected with vehicle (F2, 60 = 12.302; P = 0.002 by 2-way repeated-measures ANOVA); however, the concentration of noradrenaline was not increased by subcutaneous injection of capsaicin in rats 6 weeks after SNL surgery (Figure 4).
Effect of Amitriptyline and Pregabalin in SNL-Induced Hyperalgesia
To determine the effect of chronic treatment with amitriptyline or pregabalin on NSIA after SNL, the effects of 5 daily intraperitoneal injections of both drugs on hyperalgesia induced by SNL were evaluated. Withdrawal thresholds significantly decreased compared with the preoperative level in the ipsilateral hind paw (right) 5 weeks after SNL surgery. The amitriptyline (10 mg/kg/d) and pregabalin (10 mg/kg/d) injections, but not injection of saline, produced antihyperalgesic effects in the ipsilateral paw from day 1 (amitriptyline) or day 2 (pregabalin) after the daily injections were initiated (Figure 5, A and B, P < 0.001 by Student t test with Bonferroni correction after 2-way repeated-measures ANOVA).
Change in NSIA After Amitriptyline and Pregabalin Administration
After 5 daily injections of amitriptyline or pregabalin, the withdrawal threshold in the contralateral hind paw (left) was measured after subcutaneous injection of capsaicin in the forepaw at 6 weeks after SNL surgery. Compared with the pregabalin- and saline-treated groups, amitriptyline significantly elevated the withdrawal threshold of the left hind paw (Figure 6; F2,60 = 73.366; P < 0.001 by 2-way repeated-measures ANOVA). Thus, 5 daily injections of amitriptyline reversed the attenuation of NSIA by SNL.
Spinal Noradrenaline Release After Amitriptyline and Pregabalin Administration
The concentration of noradrenalin after forepaw capsaicin injection in the spinal cord was significantly increased by 5 daily injections of amitriptyline compared with the saline-treated group (Figure 7, F2, 60 = 9.468; P = 0.005 by 2-way repeated-measures ANOVA). In contrast, the concentration of noradrenaline after forepaw capsaicin injection in the spinal cord did not increase after 5 daily injections of pregabalin compared with the saline-treated group.
In the present study, the strength of NSIA in rats dramatically decreased from 5 weeks after SNL surgery, and this decrease occurred concurrently with a decrease in spinal noradrenaline release. Although it is not clear that the doses of drugs that we used are clinically relevant, 5 daily injections of amitriptyline and pregabalin (10 mg/kg/d) from 5 weeks after SNL effectively decreased hyperalgesia after SNL. The decrease in NSIA and noradrenaline levels, however, was reversed only by amitriptyline and not by pregabalin.
Peripheral and central mechanisms are involved in endogenous analgesia.7,21,22 Previous studies have suggested that primary afferent nociceptive input is necessary for induction of NSIA6 and that NSIA is mediated by circuits in the nucleus accumbens,6,23,24 the periaqueductal gray-rostral ventromedial medulla system,25 and the spinal cord.26–28 Glutamate receptors26,28 and inhibitory receptors such as γ-aminobutyric acid (GABA) type A, GABA type B, μ-opioid receptors27 are known to be involved in the mechanism of NSIA. A recent study also suggested that the noradrenergic system plays an important role in NSIA, as NSIA was partially blocked by intrathecal injection of an α2-adrenergic receptor antagonist and by ablation of descending noradrenergic innervation of the spinal cord.5
A previous study demonstrated plastic changes in descending noradrenergic neurons after nerve injury, where the density of the descending noradrenergic fibers and noradrenaline content in the ipsilateral lumbar spinal cord were increased 10 days after SNL in rats.10 Another report indicated a loss of noradrenergic fibers in the ipsilateral lumbar spinal cord 19 to 21 days after tibial nerve transection in rats.11 Although the animal models were different, these results suggest that the tone of the descending noradrenergic system dynamically changes over time after nerve injury.
In the present study, we therefore examined the changes in NSIA and noradrenaline release in the spinal cord after forepaw capsaicin injection in rats with SNL. We demonstrated that NSIA was strongly decreased in rats at 5 and 6 weeks compared with 4 weeks after SNL. In addition, spinal noradrenaline levels in response to forepaw capsaicin injection were not increased in rats at 6 weeks after SNL compared with normal rats. These results suggest that the attenuation of NSIA resulted at least partly from plastic changes in descending noradrenergic neurons of the locus coeruleus (LC).
Amitriptyline and pregabalin have been shown to be effective analgesics in patients with neuropathic pain,13 and both drugs may activate or enhance the descending noradrenergic inhibitory system.15,17 We suspected that the effect of these drugs against neuropathic pain might at least partly be mediated through enhanced NSIA. In the present study, 5 daily injections of amitriptyline (10 mg/kg/d) and pregabalin (10 mg/kg/d) initiated 5 weeks after SNL increased the withdrawal threshold in the ipsilateral hindpaw, indicating that both drugs were effective for SNL-induced hyperalgesia. At 6 weeks after SNL, however, the effects of the 2 drugs on NSIA were different, as amitriptyline but not pregabalin reversed the attenuation of NSIA by SNL surgery. Microdialysis studies also demonstrated that 5 daily injections of amitriptyline, but not pregabalin, increased the noradrenaline level in the spinal cord after forepaw capsaicin injection. These results suggest that amitriptyline, but not pregabalin, reversed the dysfunction of the descending noradrenergic inhibitory system after SNL.
Amitriptyline may act not only as a 5-hydroxytryptamine and noradrenaline reuptake inhibitor but also as a blocker of voltage-gated sodium channels and N-methyl-D-aspartate receptors.29 Specifically with regard to noradrenaline, a microdialysis study showed that amitriptyline strongly increased the noradrenaline level in the spinal cord.17 Pregabalin has been shown to modulate the release of several neurotransmitters by selectively binding to the α2δ subunit of voltage-dependent calcium channels to reduce the influx of Ca2+ ions.30,31 Pregabalin and another α2δ ligand, gabapentin, are thought to activate noradrenergic neurons by reducing presynaptic GABAergic activity in the LC, thereby increasing noradrenaline release, which subsequently inhibits pain transmission via spinal α2-adrenoreceptors.15 Gabapentin has also been shown to increase the noradrenaline level in the rat spinal cord after systemic injection, as measured by microdialysis.16 Given that both amitriptyline and pregabalin activate descending noradrenergic system, the reason for the discrepancy between amitriptyline and pregabalin with regard to NSIA in the present study is not clear.
One possible mechanism for the effect of amitriptyline in the present study is the modulation of noradrenergic regulation of the LC; amitriptyline is a strong noradrenaline reuptake inhibitor17 and may thus increase the noradrenaline level in the LC, whose activation is mediated by α1, β2, and β3 adrenergic receptors.32 In contrast, activation of the LC is inhibited by α2A adrenergic receptors,33,34 and thus inhibition or blockade of α2A adrenergic receptors would be expected to increase synaptic levels of noradrenaline. A recent study demonstrated that tricyclic antidepressants including amitriptyline downregulate α2A adrenergic receptors35; therefore, in the present study, the 5 daily injections of amitriptyline might have activated the LC through decreased α2A adrenergic signaling.
Another possible mechanism is the induction of glutamatergic neurotransmission in the LC by amitriptyline. A previous study demonstrated that nerve injury increases the basal extracellular glutamate concentration in the LC,8 which may reduce noxious stimulation-evoked glutamate release, thereby diminishing α-amino-3-hydroxy-5-methyl- 4-isoxazolepropionic acid (AMPA) receptor–mediated LC activation, which is important for induction of NSIA. Another study demonstrated that amitriptyline increased brain-derived neurotrophic factor levels in astrocyte cultures.36 Brain-derived neurotrophic factor triggers AMPA receptor GluA1 phosphorylation and regulates trafficking of the AMPA receptor.37 Therefore, amitriptyline may enhance glutamatergic neurotransmission by increasing AMPA receptor levels at the membrane surface in neurons of the LC. In the present study, we did not investigate the function of the LC in response to capsaicin injection; further studies are needed to investigate the differences among drugs in the context of their effect on NSIA.
In summary, NSIA was strongly decreased, and spinal noradrenaline levels were not increased after forepaw injection of capsaicin in rats at 6 weeks after SNL. Five daily injections of amitriptyline reversed the attenuation of NSIA after SNL surgery and increased the noradrenaline level in the spinal cord in response to forepaw capsaicin injection. It is therefore possible that amitriptyline reverses the attenuation of endogenous analgesia in chronic pain states.
Name: Hiroaki Matsuoka, MD.
Contribution: This author helped conduct the study, collect data, analyze the data, and prepare the manuscript.
Name: Takashi Suto, MD, PhD.
Contribution: This author helped collect the data and prepare the manuscript.
Name: Shigeru Saito, MD, PhD.
Contribution: This author helped design the study and prepare the manuscript.
Name: Hideaki Obata, MD, PhD.
Contribution: This author helped design and conduct the study, analyze the data, and prepare the manuscript.
This manuscript was handled by: Jianren Mao, MD, PhD.
We thank Dr. Devang Thakor (PhD) for English editing.
1. Yarnitsky D, Crispel Y, Eisenberg E, Granovsky Y, Ben-Nun A, Sprecher E, Best LA, Granot M. Prediction of chronic post-operative pain: pre-operative DNIC testing identifies patients at risk. Pain 2008;138:22–8.
2. Yarnitsky D, Granot M, Nahman-Averbuch H, Khamaisi M, Granovsky Y. Conditioned pain modulation predicts duloxetine efficacy in painful diabetic neuropathy. Pain 2012;153:1193–8.
3. Lewis GN, Rice DA, McNair PJ. Conditioned pain modulation in populations with chronic pain: a systematic review and meta-analysis. J Pain 2012;13:936–44.
4. Yarnitsky D. Conditioned pain modulation (the diffuse noxious inhibitory control-like effect): its relevance for acute and chronic pain states. Curr Opin Anaesthesiol 2010;23:611–5.
5. Peters CM, Hayashida K, Suto T, Houle TT, Aschenbrenner CA, Martin TJ, Eisenach JC. Individual differences in acute pain-induced endogenous analgesia predict time to resolution of postoperative pain in the rat. Anesthesiology 2015;122:895–907.
6. Gear RW, Aley KO, Levine JD. Pain-induced analgesia mediated by mesolimbic reward circuits. J Neurosci 1999;19:7175–81.
7. Tambeli CH, Levine JD, Gear RW. Centralization of noxious stimulus-induced analgesia (NSIA) is related to activity at inhibitory synapses in the spinal cord. Pain 2009;143:228–32.
8. Kimura M, Suto T, Morado-Urbina CE, Peters CM, Eisenach JC, Hayashida K. Impaired Pain-evoked Analgesia after Nerve Injury in Rats Reflects Altered Glutamate Regulation in the Locus Coeruleus. Anesthesiology 2015;123:899–908.
9. Millan MJ. Descending control of pain. Prog Neurobiol 2002;66:355–474.
10. Hayashida K, Clayton BA, Johnson JE, Eisenach JC. Brain derived nerve growth factor induces spinal noradrenergic fiber sprouting and enhances clonidine analgesia following nerve injury in rats. Pain 2008;136:348–55.
11. Hughes SW, Hickey L, Hulse RP, Lumb BM, Pickering AE. Endogenous analgesic action of the pontospinal noradrenergic system spatially restricts and temporally delays the progression of neuropathic pain following tibial nerve injury. Pain 2013;154:1680–90.
12. Hoshino H, Obata H, Nakajima K, Mieda R, Saito S. The antihyperalgesic effects of intrathecal bupropion, a dopamine and noradrenaline reuptake inhibitor, in a rat model of neuropathic pain. Anesth Analg 2015;120:460–6.
13. Finnerup NB, Attal N, Haroutounian S, McNicol E, Baron R, Dworkin RH, Gilron I, Haanpää M, Hansson P, Jensen TS, Kamerman PR, Lund K, Moore A, Raja SN, Rice AS, Rowbotham M, Sena E, Siddall P, Smith BH, Wallace M. Pharmacotherapy for neuropathic pain in adults: a systematic review and meta-analysis. Lancet Neurol 2015;14:162–73.
14. Tanabe M, Takasu K, Kasuya N, Shimizu S, Honda M, Ono H. Role of descending noradrenergic system and spinal alpha2-adrenergic receptors in the effects of gabapentin on thermal and mechanical nociception after partial nerve injury in the mouse. Br J Pharmacol 2005;144:703–14.
15. 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.
16. Hayashida K, Obata H, Nakajima K, Eisenach JC. Gabapentin acts within the locus coeruleus to alleviate neuropathic pain. Anesthesiology 2008;109:1077–84.
17. Hoshino H, Obata H, Saito S. Antihyperalgesic effect of duloxetine and amitriptyline in rats after peripheral nerve injury: Influence of descending noradrenergic plasticity. Neurosci Lett 2015;602:62–7.
18. Kim SH, Chung JM. An experimental model for peripheral neuropathy produced by segmental spinal nerve ligation in the rat. Pain 1992;50:355–63.
19. Randall LO, Selitto JJ. A method for measurement of analgesic activity on inflamed tissue. Arch Int Pharmacodyn Ther 1957;111:409–19.
20. Obata H, Kimura M, Nakajima K, Tobe M, Nishikawa K, Saito S. Monoamine-dependent, opioid-independent antihypersensitivity effects of intrathecally administered milnacipran, a serotonin noradrenaline reuptake inhibitor, in a postoperative pain model in rats. J Pharmacol Exp Ther 2010;334:1059–65.
21. Ferrari LF, Gear RW, Levine JD. Attenuation of activity in an endogenous analgesia circuit by ongoing pain in the rat. J Neurosci 2010;30:13699–706.
22. van Wijk G, Veldhuijzen DS. Perspective on diffuse noxious inhibitory controls as a model of endogenous pain modulation in clinical pain syndromes. J Pain 2010;11:408–19.
23. Schmidt BL, Tambeli CH, Barletta J, Luo L, Green P, Levine JD, Gear RW. Altered nucleus accumbens circuitry mediates pain-induced antinociception in morphine-tolerant rats. J Neurosci 2002;22:6773–80.
24. Schmidt BL, Tambeli CH, Levine JD, Gear RW. mu/delta Cooperativity and opposing kappa-opioid effects in nucleus accumbens-mediated antinociception in the rat. Eur J Neurosci 2002;15:861–8.
25. Gear RW, Levine JD. Rostral ventral medulla cholinergic mechanism in pain-induced analgesia. Neurosci Lett 2009;464:170–2.
26. Tambeli CH, Parada CA, Levine JD, Gear RW. Inhibition of tonic spinal glutamatergic activity induces antinociception in the rat. Eur J Neurosci 2002;16:1547–53.
27. Tambeli CH, Quang P, Levine JD, Gear RW. Contribution of spinal inhibitory receptors in heterosegmental antinociception induced by noxious stimulation. Eur J Neurosci 2003;18:2999–3006.
28. Tambeli CH, Young A, Levine JD, Gear RW. Contribution of spinal glutamatergic mechanisms in heterosegmental antinociception induced by noxious stimulation. Pain 2003;106:173–9.
29. Sawynok J, Esser MJ, Reid AR. Antidepressants as analgesics: an overview of central and peripheral mechanisms of action. J Psychiatry Neurosci 2001;26:21–9.
30. Dolphin AC. Calcium channel auxiliary α2δ and β subunits: trafficking and one step beyond. Nat Rev Neurosci 2012;13:542–55.
31. Li Z, Taylor CP, Weber M, Piechan J, Prior F, Bian F, Cui M, Hoffman D, Donevan S. Pregabalin is a potent and selective ligand for α(2)δ-1 and α(2)δ-2 calcium channel subunits. Eur J Pharmacol 2011;667:80–90.
32. Gibbs ME, Hutchinson DS, Summers RJ. Noradrenaline release in the locus coeruleus modulates memory formation and consolidation; roles for α- and β-adrenergic receptors. Neuroscience 2010;170:1209–22.
33. Callado LF, Stamford JA. Alpha2A- but not alpha2B/C-adrenoceptors modulate noradrenaline release in rat locus coeruleus: voltammetric data. Eur J Pharmacol 1999;366:35–9.
34. Starke K. Presynaptic autoreceptors in the third decade: focus on alpha2-adrenoceptors. J Neurochem 2001;78:685–93.
35. Cottingham C, Percival S, Birky T, Wang Q. Tricyclic antidepressants exhibit variable pharmacological profiles at the α(2A) adrenergic receptor. Biochem Biophys Res Commun 2014;451:461–6.
36. Kajitani N, Hisaoka-Nakashima K, Morioka N, Okada-Tsuchioka M, Kaneko M, Kasai M, Shibasaki C, Nakata Y, Takebayashi M. Antidepressant acts on astrocytes leading to an increase in the expression of neurotrophic/growth factors: differential regulation of FGF-2 by noradrenaline. PLoS One 2012;7:e51197.
37. Reimers JM, Loweth JA, Wolf ME. BDNF contributes to both rapid and homeostatic alterations in AMPA receptor surface expression in nucleus accumbens medium spiny neurons. Eur J Neurosci 2014;39:1159–69.