Effects of the endocannabinoid transport inhibitors AM404 and UCM707 on diabetic neuropathy in rats

• 1 Diabetic rats display increased pain responses following injection of formalin into the paw, suggesting the presence of hyperalgesia. In the present study, we investigated the efficacy of the systemic administration of the endocannabinoid transport inhibitors UCM707 and AM404 (1, 10 and 50 mg/kg, i.p.) on hyperalgesia during the formalin test in streptozocin (STZ)‐induced diabetic rats.
• 2 Nociceptive testing was performed in male adult Wistar rats 4 weeks after the onset of hyperglycaemia. At the end of the experiment, all rats were weighed and then underwent plasma glucose measurements.
• 3 Diabetes caused significant hyperalgesia during both phases of the formalin test. At 10 and 50 mg/kg, both UCM707 and AM404 reversed chemical hyperalgesia in diabetic rats. UCM707 (10 and 50 mg/kg) caused less intensive nociceptive behaviour during both phases of the test, whereas AM404 (10 and 50 mg/kg) only affected pain scores during Phase 1 of the formalin test. At 1 mg, neither drug had any effect on pain behaviour in control and diabetic groups compared with their respective controls. Neither UCM707 nor AM404 had any effect on bodyweight or plasma glucose levels of treated compared with non‐treated rats at any of the doses tested.
• 4 The results of the present study indicate that systemic administration of UCM707 and AM404 is effective in ameliorating chemical hyperalgesia in STZ‐diabetic rats. Thus, endocannabinoid transport inhibitors may have potential in the treatment of painful diabetic neuropathy.

     

贝前列素钠对糖尿病大鼠坐骨神经病变的保护作用

目的:观察贝前列素钠对糖尿病大鼠的坐骨神经病变的影响。方法:将30只SD大鼠随机分为正常对照组、模型对照组和治疗组,每组10只。模型对照组和治疗组大鼠腹腔注射链佐星60mg/kg建立糖尿病模型,24周时测定3组大鼠的运动神经传导速度(motor nerve conduction velocity,MNCV)和感觉神经传导速度(sensory nerve conduction velocity,SNCV),用光学显微镜和电镜观察大鼠的坐骨神经切片,比较其病理改变。结果:建模后24周时,模型对照组大鼠与正常对照组大鼠相比,神经传导速度下降[MNCV(3.83±0.22)vs(4.59±0.87)m/s(P<0.01);SNCV(6.25±0.37)vs(9.26±0.53)m/s(P<0.01)],坐骨神经发生明显病理性改变。治疗组大鼠与模型对照组大鼠比较,神经传导速度显著升高[MNCV(6.83±0.86)vs(3.83±0.22)m/s(P<0.01);SNCV(7.32±0.78)vs(6.25±0.37)m/s(P<0.05)],坐骨神经病变程度有明显改善。结论:贝前列素钠可增加糖尿病大鼠的坐骨神经传导速度,减轻坐骨神经病变程度。     

Alleviation of pain in painful diabetic neuropathy

Introduction: Painful diabetic neuropathy (PDN) is a disabling pain condition. Its pathomechanism remains unknown, but a sensitization and neuronal hyperexcitabilty have been suggested. Only symptomatic pharmacological pain management treatment is currently available.

Areas covered: The origin of PDN is enigmatic, and the evidence-based therapeutic guidelines therefore consist only of antidepressants and antiepileptics as first-line recommended drugs. This article relates to a MEDLINE/PubMed systematic search (2005-2015).

Expert opinion: The results of the meta-analysis from the aspect of the efficacy of amitriptyline, duloxetine, venlafaxine, gabapentin and pregabalin are favorable, but the placebo response rate is relatively high in patients with neuropathic pain. For personalization of the medication of PDN patients, the optimum dosing, the genotyping of the metabolizing enzymes and optimum biomarkers are needed. As concerns the future perspectives, specific sodium channel subtype inhibitors acting on peripheral nociceptive neurons or modified T-type voltage-gated calcium channel blockers may be promising targets for pharmaceutical innovations. Another attractive strategy for the treatment is based on the effects of monoclonal antibodies against nerve growth factor, sodium channels, specific receptor and cytokines. Botulinum toxin A, capsaicin patch and spinal cord stimulation therapies are the nearest future therapeutic options for the treatment of PDN patients.     

Effect of rosmarinic acid on experimental diabetic nephropathy

Connective tissue growth factor (CTGF) plays a pathogenic role in diabetic nephropathy (DN). Rosmarinic acid (RA) is a naturally occurring phenolic acid. This study was conducted to investigate the efficacy of RA on DN and to elucidate the potential mechanism. High glucose (HG)-stimulated cultured human renal proximal tubular epithelial cells (HK-2) analysed CTGF expression by western blotting, and it was investigated whether extracellular signal-regulated kinase (ERK) signalling pathway was involved. Using streptozotocin (STZ)-induced rat animal models, diabetic rats were randomized to receive intragastric (i.g.) doses of RA. Renal tissue, blood and urine samples were collected to determine biochemical index and analyse protein expression. In vitro study, RA reduced CTGF excretion in HG-induced HK-2 cells through the ERK signalling pathway. In an in vivo study, I.g. of RA 7.5 or 15 mg/kg significantly ameliorated renal function and increased body-weight. Meanwhile, RA reduced renal CTGF expression by immunohistochemical staining and reduced serum levels of CTGF. Besides, there were no significant differences in glycaemia levels between the RA groups compared with the STZ-treated group. Furthermore, RA ameliorated renal pathology. These results suggest that RA exerts an early renal protective role to DN. Inhibition of CTGF may be a potential target in DN therapy, which highlights the possibility of using RA in the treatment of DN.     

自发性糖尿病GK 大鼠糖尿病周围神经病变模型的建立

摘要： 目的 通过高脂饲养 GK 大鼠建立一种符合人类糖尿病周围神经病变 （DPN） 发病特点, 又操作简单的 DPN 大鼠模型。方法 7~8 周龄 SPF 级雄性自发性 2 型糖尿病 GK 大鼠 30 只, 喂养高脂饲料造模。另取 30 只 SPF 级正常 Wistar 大鼠作为正常组, 喂养普通饲料。每周监测动物血糖、 体质量、 饮水量、 饲料量。分别于干预 8 周、 12 周、 16 周后检测血清糖化血红蛋白、 坐骨神经传导速度, 取对侧坐骨神经做 HE 染色, TUNEL 染色计算细胞凋亡指数。结果 随着造模时间延长, GK 大鼠逐渐出现多饮、 多食、 生长迟缓等表现。与正常组相比, 造模 12 周及 16 周的大鼠血糖、 糖化血红蛋白升高 （P < 0.01）, 感觉神经传导速度降低 （P < 0.01）, 运动神经传导速度有一定下降趋势（12 周 P < 0.05, 16 周 P > 0.05）, 坐骨神经病理形态及雪旺细胞凋亡情况均提示 DPN 的形成, 并与正常大鼠形成鲜明对照 （凋亡指数 P < 0.01）。结论 长期高脂饲料喂养的 GK 大鼠是 DPN 研究的良好模型, 12 周是较为成熟且经济的造模时间。     

Effects of trolox on nerve dysfunction, thermal hyperalgesia and oxidative stress in experimental diabetic neuropathy

1. Diabetic neuropathy is one of the most common complications of diabetes and oxidative stress has been implicated to play a major role in its pathophysiology. 2. In the present study, we targeted oxidative stress using trolox, an anti-oxidant, in streptozotocin-induced diabetic neuropathy in rats. 3. Compared with control rats, diabetic rats showed significant deficits in motor nerve conduction velocity (MNCV; 49.91 +/- 1.94 vs 42.77 +/- 1.39 m/s, respectively) and nerve blood flow (NBF; 107.98 +/- 8.22 vs 38.9 +/- 2.7 arbitarary perfusion units, respectively) after 8 weeks of diabetes. Tail flick latencies for cold and hot immersion tests were also significantly reduced in diabetic rats, indicating thermal hyperalgesia. These observations indicate development of diabetic neuropathy. 4. A significant decrease in the activity of anti-oxidant enzymes (superoxide dismutase and catalase) and an increase in lipid peroxidation were observed in sciatic nerves from diabetic rats compared with age-matched control rats. Alterations in the activity of anti-oxidant enzymes and lipid peroxidation in diabetic rats indicate oxidative stress in diabetic neuropathy. 5. Two weeks treatment with trolox (10 and 30 mg/kg, i.p.) started on completion of the 6th week of diabetes significantly improved MNCV, NBF and inhibited thermal hyperalgesia. Trolox treatment also improved the activity of anti-oxidant enzymes and inhibited lipid peroxidation in sciatic nerves of diabetic rats. 6. The results of the present study suggest the beneficial effects of trolox in experimental diabetic neuropathy.     

Therapeutic potential of PKC inhibitors in painful diabetic neuropathy

Diabetic neuropathy accompanied by anomalies in pain perception is one of the most frequent complications in insulin-dependent diabetes in humans. Many clinical and experimental studies have suggested that diabetes or hyperglycaemia alters pain sensitivity. In humans, diabetic neuropathy can be associated with burning, tactile hypersensitivity. Behavioural reactions of hyperalgesia in animal models of diabetes have been described. However, the aetiology of these disturbances is still unknown, although metabolic factors such as hyperglycaemia or neurotransmitter alteration may be involved. Activation of protein kinase C (PKC) has been implicated in changes in pain perception. Phorbol esters, which activate PKC, enhance the thermal hyperalgesia in diabetic mice and enhance nociceptive responses after tissue injury induced by formalin. Electrophysiological experiments have shown that activation of PKC leads to long-lasting enhancement of excitatory amino acid-mediated currents in dorsal horn neurons and trigeminal neurons. Thus, activation of PKC may underlie the neuronal sensitisation that produces hyperalgesia in diabetic neuropathy.     

FK1706, a novel non-immunosuppressive immunophilin ligand, modifies the course of painful diabetic neuropathy

FK1706, a derivative of FK506, is a non-immunosuppressive immunophilin ligand with significant neurotrophic activity mediated via FKBP-52 and the RAS/RAF/MAPK signaling pathway. Here, we tested the effect of FK1706 on painful diabetic neuropathy in rat model of diabetes induced by streptozotocin (STZ). FK1706 ameliorated mechanical allodynia in this model at doses over 0.32 mg/kg, p.o., even if treatment was initiated after neuropathy was established, and did not affect plasma glucose levels. Furthermore, this improvement continued at least 4 weeks after the last administration. In morphological analysis, FK1706 treatment also restored intraepidermal nerve fiber density in footpad skin to almost normal levels. Gabapentin also improved mechanical allodynia in the same model, but efficacy disappeared the day after administration stopped. Allodynia responses were potentiated by co-administration of both compounds. Thus, FK1706 ameliorated painful diabetic neuropathy via a different mechanism from gabapentin and improved morphological outcomes, indicating that FK1706 improves painful diabetic neuropathy by modifying the underlying disease pathology.     

A spinal mechanism of action for duloxetine in a rat model of painful diabetic neuropathy

BACKGROUND AND PURPOSE

This study was designed to clarify mechanisms responsible for the anti-allodynic effects of duloxetine in diabetes.

EXPERIMENTAL APPROACH

The streptozotocin-induced diabetic rat model was used to compare the efficacy of duloxetine, 5-HT, the 5-HT_2A receptor agonist [1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane hydrochloride (DOI)] and two antagonists (ketanserin and pruvanserin) on tactile allodynia.

KEY RESULTS

Systemic or intrathecal injection of duloxetine alleviated tactile allodynia in diabetic rats. The effect of systemic duloxetine was reduced by intrathecal administration of ketanserin or pruvanserin, indicating participation of spinal 5-HT_2A receptors in the mechanism of action of duloxetine. In contrast to spinal delivery, systemic and local peripheral injections of ketanserin or pruvanserin alleviated tactile allodynia in diabetic rats. This effect was reversed immediately after systemic or local DOI injection.

CONCLUSIONS AND IMPLICATIONS

These results support the involvement of spinal 5-HT_2A receptors in the ability of duloxetine to ameliorate painful diabetic neuropathy. Our data also suggest that the role of 5-HT_2A receptors depends on the level of the neuraxis at which activation takes place, with peripheral activation contributing to tactile allodynia in diabetic rats, whereas spinal activation of this receptor alleviates tactile allodynia. The development of selective peripheral 5-HT_2A receptor antagonists may offer a novel approach for the treatment of diabetic neuropathic pain.     

中药仙灵脾对2型糖尿病大鼠炎症因子的影响

目的:评价中药仙灵脾对2型糖尿病大鼠炎症因子的影响。方法:雄性Wistar大鼠70只,从中随机选取10只为正常对照组,其余应用高糖高脂饲料喂养及链脲佐菌素腹腔注射造模,成模2型糖尿病大鼠共44只按血糖值随机分组:糖尿病(DM)模型对照组(16只)、仙灵脾组(16只)、二甲双胍组(12只),各组共相应给药8周,治疗前后取血测量血糖(FBG)、C反应蛋白(CRP)、肿瘤坏死因子-α(TNF-α)、白细胞介素6(IL-6)。结果:成模2型糖尿病各组大鼠FBG,CRP,TNF-α,IL-6水平均较正常对照组大鼠明显增高(P<0.01)。应用仙灵脾及二甲双胍治疗的两组大鼠的FBG,CRP,TNF-α,IL-6水平均较DM模型对照组显著下降(P<0.05),但两治疗组比较差别无统计学意义(P>0.05)。结论:中药仙灵脾治疗2型糖尿病大鼠可降低炎症因子水平,抑制体内炎症反应。     

Altered antinociceptive efficacy of tramadol over time in rats with painful peripheral neuropathy

Pain due to peripheral nerve injury or disease is a dynamic process, such that the mechanism that underlies it alters over time. Tramadol has been reported to be analgesic in clinical neuropathic pain, with varying levels of efficacy due to a patient population that has had neuropathic pain for a wide range of time. In order to address and examine the issue, the antinociceptive efficacy of tramadol over time was tested in rats with a chronic constriction injury (CCI) of the left sciatic nerve. Rats developed a robust hind paw hypersensitivity to innocuous mechanical stimulation ipsilateral to CCI surgery. Subcutaneous injection of tramadol in rats two weeks after CCI surgery dose-dependently attenuated mechanical hypersensitivity, which was abolished with the mu-opioid receptor antagonist naloxone but not the alpha(2)-adrenoceptor antagonist yohimbine. Systemic tramadol also attenuated mechanical hypersensitivity four weeks after CCI surgery, but the efficacy significantly diminished at this time point. In addition, the effect of tramadol at this later time point could be reduced with yohimbine as well as naloxone. These data demonstrate that the efficacy of tramadol depends in part on the duration of nerve injury-evoked nociception, and that its antinociceptive mechanism changes over time. Alteration in antinociceptive mechanism over time may explain the inconsistency in efficacy of this and other analgesic drugs in chronic pain patients.     

鞘内注射氟代柠檬酸对糖尿病大鼠神经病理性疼痛的影响

研究发现脊髓的星形胶质细胞在疼痛的发生和维持中起到十分重要作用[1～3].氟代柠檬酸(flourocltrate,FC)是星形胶质细胞的特异性抑制剂,它在脊神经损伤模型中能抑制星形胶质细胞过渡活化,减轻神经病理性疼痛[2,3].但氟代柠檬酸对糖尿病大鼠神经病理性疼痛的影响如何尚未见研究.本研究观察氟代柠檬酸对糖尿病大鼠神经病理性疼痛模型大鼠痛行为及脊髓星形胶质细胞的影响,以探讨其机制.     

吗啡对糖尿病神经病理痛大鼠机械及冷刺激痛敏症状的影响

目的探讨吗啡对糖尿病神经病理痛大鼠机械及冷刺激痛敏症状的影响。方法35只SD雄性大鼠(180～220 g)腹腔注射链脲佐菌素(STZ)制备成糖尿病神经病理痛模型,出现痛敏症状后,随机分为5组,采用von Frey测试针和冷板(5度)法,测量鞘内给予吗啡后不同时间机械缩腿阈值和冷刺激后的抬足次数(次/5min)变化。结果吗啡以剂量依赖方式提高糖尿病神经痛大鼠机械缩腿阈值、降低冷刺激后的抬足次数,给予吗啡后30 min,0.5、1.0、5.0μg剂量吗啡组的机械缩腿阈值明显增高,而冷刺激抬足次数分别为显著下降,分别与用药前和对照组相比,差异有统计学意义(P<0.05)。结论吗啡能显著提高糖尿病神经痛大鼠的机械缩腿阈值,有效减少冷刺激的抬足次数,对神经源性痛具有明显的镇痛作用。     

Effects of pregabalin on heart rate variability in patients with painful diabetic neuropathy

Many studies have demonstrated that low heart rate variability (HRV) is a risk for high mortality and morbidity in patients with cardiovascular diseases. The primary purpose of the study was to evaluate whether pregabalin improves HRV in patients with diabetes and painful peripheral neuropathy. Resting heart rates were collected by using the LifeShirt System, developed by VivoMetrics (Ventura, Calif), at baseline and at the end of a 4-week intervention of pregabalin or placebo in patients with painful diabetic peripheral neuropathy. Heart rate variability analysis was performed on the collected R-R intervals using the Vivo- VMLA-036-00 3 Logic of the LifeShirt system. Of the 40 patients enrolled in the study, 70% completed the end of 4-week assessments (n = 15 in pregabalin and n = 14 in placebo). Compared with placebo, pregabalin treatment resulted in significant improvement in HRV measured by frequency domain analysis, that is, a reduction in low frequency-high frequency ratio (-1.30 ± 2.89 vs 0.37 ± 0.33, P = 0.03) and power of normalized low frequency (-0.049 ± 0.092 vs 0.0066 ± 0.023, P = 0.02), as well as an increase in power of normalized high frequency (0.039 ± 0.094 vs -0.038 ± 0.066, P = 0.02). Furthermore, pregabalin resulted in greater reduction of pain and symptoms of anxiety and greater improvement of quality of life. The improvement of HRV measures were not correlated with change of those measures. In conclusion, 4-week pregabalin treatment improved HRV in patients with painful diabetic peripheral neuropathy. Trial Registration: NCT00573261 (clinicaltrials.gov).     

Effects of rosmarinic acid on acetaminophen-induced hepatotoxicity in male Wistar rats

Context: Drug-induced liver injury is a significant worldwide clinical problem. Rosmarinic acid (RA), a natural phenol, has antioxidant effects.

Objective: The effects of RA against acetaminophen (N-acetyl-p-amino-phenol (APAP))-induced oxidative damage and hepatotoxicity in rats were investigated.

Materials and methods: Male Wistar rats were pretreated with RA (10, 50 and 100?mg/kg, i.g.) for one week. On day 7, rats received APAP (500?mg/kg, i.p.). Then aspartate aminotransferase (AST), alanine aminotransferase (ALT), albumin, total protein, malondialdehyde (MDA), glutathione (GSH), total antioxidant capacity (TAC), glutathione S-transferase (GST), cytochrome CYP450 and histopathological changes were determined.

Results: APAP-induced oxidative stress in liver by a significant increase in the level of MDA (7.6?±?0.21?nmol/mg) as well as a decrease in the contents of TAC (1.75?±?0.14?μmol/g), GSH (1.9?±?0.22?μmol/g) and GST) 3.2?±?0.28?U/mg). RA treatment decreased MDA (4.32?±?0.35?nmol/mg) but increased the contents of TAC (3.51?±?0.34?μmol/g), GSH (3.42?±?0.16?μmol/g) and GST (5.71?±?0.71?μmol/g) in APAP group. RA 100?mg/kg decreased ALT (91.5?±?1.5?U/L), AST (169?±?8.8?U/L) and CYP450 (3?±?0.2?nmol/min/mg) in APAP group. Histologically RA attenuated hepatic damage by decreasing necrosis, inflammation, and haemorrhage in liver sections of APAP group.

Discussion and conclusions: This is the first report that oral administration of RA dose-dependently elicited significant hepatoprotective effects in rats through inhibition of hepatic CYP2E1 activity and lipid peroxidation. RA-protected hepatic GSH and GST reserves and total tissue antioxidant capacity.     

Antinociceptive efficacy of lacosamide in a rat model for painful diabetic neuropathy

Lacosamide was tested in the streptozotocin rat model of diabetic neuropathic pain in comparison to drugs which are commonly used in the treatment of diabetic neuropathic pain, i.e. antidepressants and anticonvulsants. In diabetic rats, lacosamide attenuated cold (10, 30 mg/kg, i.p.), warm (3, 10, 30 mg/kg, i.p.) and mechanical allodynia (30 mg/kg, i.p.). Streptozotocin-induced thermal and mechanical hyperalgesia were reduced by lacosamide at doses of 10 and 30 mg/kg, i.p. Morphine (3 mg/kg) showed similar efficacy on allodynia and hyperalgesia. Amitriptyline (10 mg/kg), venlafaxine (15 mg/kg), levetiracetam (180 mg/kg) and pregabalin (100 mg/kg) exhibited significant effects on thermal allodynia and mechanical hyperalgesia. Only treatment with amitriptyline (30 mg/kg, i.p.) produced full reversal of thermal allodynia comparable to lacosamide. Lamotrigine (45 mg/kg, i.p.) had no effect on both behavioral readouts. Lacosamide's potency and efficacy in reversing pain behavior might be due to its new, yet unknown mechanism of action.     

Effects of lisinopril on streptozotocin-induced diabetic neuropathy in rats

Streptozotocin (STZ)-induced diabetic neuropathy in rats was monitored by measuring the motor nerve conduction velocity (MNCV) and histopathology of the tibial nerve. Pretreatment with lisinopril (2 mg/kg p.o., 5 days prior to STZ and continued for 10 weeks) significantly (p < 0.01) prevented deterioration of MNCV as compared to STZ-diabetic animals. Nerve sections from the lisinopril pretreated group revealed less structural damage as compared to STZ-diabetic rats. However lisinopril had no effect on blood sugar, i.e., it did not alter the diabetic state. It is concluded that lisinopril prevents the development of experimental diabetic neuropathy in STZ-induced diabetic rats.     

Effects of subcutaneous and intracerebroventricular injection of physostigmine on the acute corneal nociception in rats

The present study investigated the effects of subcutaneous (sc) and intracerebroventricular (icv) injections of physostigmine (a cholinesterase inhibitor), atropine (an antagonist of muscarinic cholinergic receptors) and hexamethonium (an antagonist of nicotinic cholinergic receptors) on the acute corneal nociception in rats. Local application of 5 M NaCl solution on the corneal surface of the eye produced a significant nociceptive behavior, characterized by eye wiping. The number of eye wipes was counted during the first 30 s. The sc (0.25, 0.5 and 1 mg/kg) and icv (1.25, 2.5, 5 and 10 μg) injections of physostigmine significantly (p < 0.05) decreased the number of eye wipes. Atropine and hexamethonium at (2 mg/kg, sc and 20 μg, icv) had no effects when used alone, however, atropine, but not hexamethonium prevented the antinociception induced by physostigmine (sc and icv). The results of this study indicate that the central muscarinic, but not nicotinic receptors might be involved in the antinociceptive effect of physostigmine in the acute corneal model of pain in rats.     

Evidence for adenosine- and serotonin-mediated antihyperalgesic effects of cizolirtine in rats suffering from diabetic neuropathy

Cizolirtine is a novel non-opioid drug which demonstrated antinociceptive activity in numerous pain models in rodents. Yet, its mechanism of action remains unknown. Several lines of evidence support the idea that adenosine (ADO) and serotonin (5-HT) modulate nociceptive signaling. Our study aimed at investigating whether these neuroactive molecules could be implicated in the mechanism of action of cizolirtine. Cizolirtine-induced antihyperalgesia was compared before and after pretreatment with ADO A(1)-A(2A) and 5-HT(1B/1D) receptor ligands in rats rendered diabetic by streptozotocin pretreatment and suffering from neuropathic pain. Cizolirtine alone (30-80 mg/kg, i.p.) significantly increased mechanical nociceptive thresholds. Acute pretreatment with the A(1)-A(2A) receptor antagonist caffeine (5 mg/kg, i.p.) or the 5-HT(1B/1D) receptor antagonist GR-127,935 (3 mg/kg, i.p.) significantly reduced the antihyperalgesic effects of cizolirtine. Conversely, cizolirtine-induced antihyperalgesia was promoted by pretreatment with either the selective A(1) receptor agonist CPA (0.3 mg/kg, i.p.) or the selective 5-HT(1B) receptor agonist CP-94,253 (3mg/kg, i.p.), and this potentiation was totally prevented by acute pretreatment with respective antagonists. Interestingly, A(1) receptor blockade by DPCPX inhibited the promoting effect of CP-94,253 on cizolirtine-induced antihyperalgesia, suggesting that the adenosine A(1)-mediated step takes place downstream the serotonin 5-HT(1B)-mediated step in the neurobiological mechanisms underlying cizolirtine action.