The effect of intra-cerebroventricular injection of insulin on the levels of monoamines on the raphe magnus nucleus of non-diabetic and short-term diabetic rats in the formalin test

Document Type : Original Article


1 Department of Basic Sciences, School of Veterinary Medicine, Shiraz University, Shiraz, Iran

2 Department of Biochemistry, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran


Objective(s): Systemic and intracerebroventricular (ICV) injection of insulin possess analgesic effects. The raphe magnus nucleus (RMN) is part of the endogenous analgesia system. The objective of the present study was to evaluate the effects of ICV injection of insulin on the levels of monoamines and their related metabolites in the RMN during the formalin test in non-diabetic and short-term diabetic rats.
Materials and Methods: Sixty four adult male rats were used. Diabetes was induced by Streptozotocin (STZ) (60 mg/kg, IP); insulin (5 mU/animal, 5 μl) was injected into the left ventricle. Microdialysis was performed in each rat. Samples were collected at 15 min intervals. After taking the base sample of microdialysis, 50 μl of 2.5% formalin was injected into the plantar surface of the hind paw, and the level of nociception was recorded every 15 sec for 1 hr. Monoamines and their metabolites concentrations were measured using the HPLC-ECD method.
Results: Findings showed that ICV injection of insulin in non-diabetic rats increased the concentration of monoamines and their related metabolites in the RMN.  In diabetic rats, injection of insulin decreased the concentrations of monoamines and their related metabolites in the RMN (P<0.5). Our results determined that, at least in part, insulin is associated with antinociceptive effect in non-diabetic rats.
Conclusion: Based on the results, it seems that ICV injection of insulin in non-diabetic rats increased the activity of the central pain control pathways leading to antinociceptive response, but this condition was not seen in diabetic rats.


Main Subjects

1. Amini-Khoei H, Amiri S, Mohammadi-Asl A, Alijanpour S, Poursaman S, Haj-Mirzaian A, Rastegar M, Mesdaghinia A, Banafshe HR, Sadeghi E, Samiei E, Mehr SE, Dehpour AR. Experiencing neonatal maternal separation increased pain sensitivity in adult male mice: involvement of oxytocinergic system. Neuropeptides 2017; 61, 77-85.
2.Hermann DM, Luppi P.H, Peyron C, Hinckel P, Jouvet M. Afferent projections to the rat nuclei raphe magnus, raphe pallidus and reticularis gigantocellularis pars demonstrated by iontophoretic application of choleratoxin (subunit b). J Chem Neuroanat 1977; 13: 1-21.
3.Millan MJ. Descending control of pain. Prog Neurobiol 2002; 66: 355-474.
4.Calvino B, Grilo Rm. Central pain control. Joint Bone Spine 2006; 73: 10- 16.
5.Hassanipour M, Amini-Khoei H, Shafaroodi H, Shirzadian A, Rahimi N, Imran-Khan M, et al. Atorvastatin attenuates the antinociceptive tolerance of morphine via nitric oxide dependent pathway in male mice. Brain Res Bull 2016; 125, 173-180.
6.Samuels ER, Szabadi E. Functional neuroanatomy of the noradrenergic locus coeruleus: its roles in the regulation of arousal and autonomic function part I: principles of functional organisation. Curr Neuropharmacol 2008; 6: 235-253.
7.Guneli E, Gumustekin M, Ates M. Possible involvement of ghrelin on pain threshold in obesity. Med Hypotheses 2010; 74: 452-454.
8.Hoang Do O, Thorn P. Insulin secretion from beta cells within intact islets: Location matters. Clin Exp Pharmacol Physiol 2015; 42: 406–414.
9.Deeds MC, Anderson JM, Armstrong AS, Gastineau DA, Hiddinga HJ, Jahangir A, Eberhardt NL, Kudva YC. Single dose streptozotocin-induced diabetes: considerations for study design in islet transplantation models. Lab Anim 2011; 45: 131-40.
10.Banks WO. JB, Erickson MA. Insulin in the brain: there and back again. Pharmacol Ther 2012; 136: 82-93.
11.Blazquez E, Velazquez E, Hurtado-Carneiro V, Ruiz-Albusac JM. Insulin in the brain: its pathophysiological implications for States related with central insulin resistance, type 2 diabetes and Alzheimer's disease. Front Endocrinol (Lausanne) 2014; 5:1- 21.
12.Duarte AI, Moreira PI, Oliveira CR. Insulin in central nervous system: more than just a peripheral hormone. J Aging Res 2012; Article ID 384017: 1-21.
13.Plum L, Schubert M, Bruning JC. The role of insulin receptor signaling in the brain. Trends Endocrinol Metab 2005; 16: 59-65.
14.Schulingkam RP, Pagano TC, Hung D, Raffa RB. Insulin receptors and insulin action in the brain: review and clinical implications. Neurosci Biobehav Rev 2000; 24: 855–872.
15.Zhao WQ, Alkon DC. Roles of the brain insulin receptor in spatial learning. Mol Cell Endocrinol 2001; 177: 125-134.
16.Hui L, Pei D, Zhang Q, Guan Q, Zhang G. The neuroprotection of insulin on ischemic brain injury in rat hippocampus through negative regulation of JNK signaling pathway by PI3K/Akt activation. Brain Research 2005; 1052: 1- 9.
17.Anuradha K, Hota D, Pandhi P. Possible mechanisms of insulin antinociception. Methods Find Exp Clin Pharmacol 2004; 26: 5-8.
18.Takeshita N, Yamaguchi I. Insulin attenuates formalin-induced nociceptive response in mice through a mechanism that is deranged by diabetes mellitus. J Pharmacol Exp Ther 1997; 28: 315-21.
19.Balali Dehkordi Sh, Sajedianfard J, Owji AA. The effect of intra-cerebroventricular injection of insulin on nociception of formalin test in non-diabetic and short-term diabetic rat models. Iran J Vet Res 2017; 18: 108- 112.
20.Ossipov MH, Dussor GO, Porreca F. Central modulation of pain. J. Clin. Investig 2010; 120: 3779–3787.
21.Davies MI, Cooper JD, Desmond S, Lunte, C, Lunte S. Analytical considerations for microdialysis sampling. Adv. Drug Deliv. Rev 2000; 45: 169– 188.
22.Gomar AHA, Mirazi N, Gomar M. Antinociceptive effect of Brassica juncea on peripheral neuropathy induced by diabetes in rat. Arak Med Uni J 2014; 17: 63-70.
23.Silva, L. Central effects of insulin and IGF1 in diabetic neuropathy. MSc Thesis, 2010. pp. 74.
24.Sharp T, Zetterstrom T. In vivo measurement of monoamine neurotransmitter release using brain microdialysis. In: Stamford, J.A. (Ed.), Monitoring Neuronal Activity: A Practical Approach. Oxford University Press, London, 1992; pp. 147–179.
25.Robert F, Lambas-Senas L, Ortemann C, Pujol JF, Renaud B. Microdialysis monitoring of 3, 4-dinydroxy phenylalanin accumulation changes in tyrosine hydroxylase activity of rat locus coeruleus. J. Neurochem 1993; 60: 721– 729.
26.Paxinos G, Watson C. The rat brain in stereotaxic coordinates. 6th ed. Sydney: Academic Press 2006; pp 112, 278.
27.Mobasher MA, Sajedianfard J, Jamshidzadeh A, Naghdi N, Namvaran MM. The effects of tramadol on norepinephrine and MHPG releasing in locus coeruleus in formalin test in rats: a brain stereotaxic study. Iran J Basic Med Sci 2014; 17: 419- 425.
28.Rahimi K, Sajedianfard J, Owji AA. The effect of intracerebroventricular injection of CGRP on pain behavioral responses and monoamines concentrations in the periaqueductal gray area in rat. Iran J Basic Med Sci 2018; 21: 395- 399.
29.Dubbuisson D, Dennis SG. The formalin test: a quantative study of the analgesic effects of morphine, meperidine and brain stem stimulation in rats and cats. Pain 1977; 4: 161-174.
30.Gordon AE, Meldrum B S. Effect of insulin on brain 5-hydroxytryptamine and 5-hydroxy-indole-acetic acid of rat. Biochem Pharmacol 1970; 19: 3042-3044.
31.Gupta G, Azam M, Baquer N Z. Effect of experimental diabetes on the catecholamine metabolism in rat brain. J Neurochem 1992; 58: 95-100.
32.Raina GS, Khurana A, Soni M. Role of thiamine and its moieties in growth rate of diatom SP. Nociceptive pain current updates in mechanisms and pathways. Int. J. Pharma Bio Sci 2011; 2: 313- 331.
33.Ennis M, Aston-Jones G, Shiekhattar R. Activation of locus coeruleus neurons by nucleus paragigantocellularis or noxious sensory stimulation is mediated by intracoerullear excitatory amino acid neurotransmission. Brain Res 1992; 598: 185– 195.
34.Singewald N, Philippu A. Release of neurotransmitters in the locus coeroleus. Prog. Neurobiol 1998; 56: 237– 267.
35.Singewald N, Zhou GY, Schneider C. Releasing of excitatory and inhibitory amino acid from the locus coeruleus of conscious rats by cardiovascular stimuli and various forms of acute stress. Brain Res 1995; 704: 42– 50.
36.Bie B, Fields HL, Williams JT, Pan ZZ. Roles of α1- and α2- adrenoceptors in the nucleus raphe magnus in opioid analgesia and opioid abstinence-induced hyperalgesia. J Neurosci 2003; 23:7950–7957.
37.Kandel E, Schwartz JH, Jessell TM, Siegelbaum SA, Hudspeth AJ. Principal of Neural Science. 5th ed.  New York City: Mc Graw Hill; 2013 p 530- 555.
38.Beitz A.J. The sites of origin of brain stem neurotensin and serotonin projections to the rodent nucleus raphe magnus. J Neurosci 1982; 2: 829- 842.
39.Uhl GR, Goodman RR, Snyder SH. Neurotensin-containing cell bodies, fibers and nerve terminals in the brain stem of the rat: Immunohistochemical mapping. Brain Res 1979; 167: 77- 91.
40.Moss MS, Glazer EJ, Basbaum AI. Enkephalin-immunoreactive perikarya in the cat raphe dorsalis. Neurosci Lett 1981; 21: 33- 37.
41.Moss MS, Glazer EJ, Basbaum AI. The peptidergic organization of the cat periaqueductal gray. I. The distribution of immunoreactive enkephalin-containing neurons and terminals. J Neurosci 1983; 3: 603-616.
42.Magoul R, Onteniente B, Oblin A, Calas A. Inter- and intracellular relationship of substance P containing neurons with serotonin and GABA in the dorsal raphe nucleus: Combination of autoradiographic and immunocytochemical techniques. J Histochem Cytochem 1986; 34: 735-742.
43.Trulson ME, Cannon MS, Raese JD. Identification of dopamine-containing cell bodies in the dorsal and median raphe nuclei of the rat brain using tyrosine hydroxylase immunochemistry. Brain Res 1985; 15:229-234.
44.Phillips S, Gelgor L, Mitchell D. Antinociceptive action of dopamine agonists in the nucleus raphe magnus of rats is mediated by D2 receptors. Pain 1992; 319: 66- 75.
45.Raz I, Hasdai D, Seltzer Z, Melmed RN. Effect of hyperglycemia on pain perception and on efficacy of morphine analgesia in rats. Diabetes 1988; 37: 1253-9.
46.Milan M, Herz A. The endocrinology of opioids. Int Rev Neurobiol 1985; 26: 1-83.
47.Kolta MG, Soliman KF, Williams BB. Role of 5-hydroxytryptamine in the regulation of brain neuropeptides in normal and diabetic rat. Horm Res 1986; 23: 112-121.
48.Iversen LL, Iversen SD, Snyder SH. Drugs, neurotransmitters and behavior. In: Handbook of Psychopharmacology, Plenum, New York; 1984 p 343- 395.
49.Sewell RS, Spencer PS. The role of biogenic amines in the action of centrally acting analgesics, in: G. W. Ellis, GG. (Ed.), Progress in medicinal chemistry. 1977; 249-283.
50.Taber RI, Latranyi MB. Antagonism of the analgesic effect of opioid and non-opioid agents by p-chlorophenylalanine (PCPA). Eur J Pharmacol 1981; 75: 215-222.
51.Sajedianfard J, Khatami S, Semnanian S, Naghdi N, Jorjani M. In vivo measurement of noradrenaline in the locus coeruleus of rats during the formalin test: a microdialysis study. Eur J Pharmacol 2005; 512: 153-156.
52.Kawahara H, Kawahara Y, Westerink BH. The noradrenaline-dopamine interaction in the rat medial prefrontal cortex studied by multi-probe microdialysis. Eur J Pharmacol 2001; 418: 177-86.
53.Fernstrom J.D. Food components to enhance performance: Stress and monoamine neurons in the brain. National Academy Press, Washington, D.C. 1994; 161- 176.
54.Inase M, Nakahama H, Otsuki T, Fang JZ. Analgesic effects of serotonin microinjection into nucleus raphe magnus and nucleus raphe dorsalis evaluated by the monosodium urate (MSU) tonic pain model in the rat. Brain-Res 1987; 426: 205-211.
55.Smith Y, Kieval JZ. Anatomy of the dopamine system in the basal ganglia. Trends Neurosci 2000; 23: 28- 33.
56.Wood PB. Role of central dopamine in pain and analgesia. Expert Review of Neurotherapeutics 2008; 8: 781-97.
57.Ohkubo Y, Nomura K, Yamaguchi I. Involvement of dopamine in the mechanism of action of FR64822, a novel non-opioid antinociceptive compound. Eur J Pharmacol 1991; 204:121-5.