Mechanisms of Plasticity of Inhibition in Chronic Pain Conditions

  • Charalampos Labrakakis
  • Francesco Ferrini
  • Yves De Koninck
Chapter

Abstract

The balance between inhibition and excitation in the dorsal spinal cord plays a critical role in ensuring that sensory information is relayed accurately to the brain. In particular, a loss of inhibitory control, and the ensuing increase in excitability in spinal dorsal horn neuronal circuits, appears to be a key substrate of pain hypersensitivity. In this Chapter, we summarize the most current knowledge on the involvement of altered GABA and glycine-mediated inhibition in pathological pain. Particular emphasis has been given to the recent finding that altered intracellular chloride homeostasis in neurons of the superficial dorsal horn may explain how inhibition is impaired following peripheral nerve injury and how this may underlie the development of neuropathic pain syndromes. Of particular interest is the finding that this mechanism of injury-induced central disinhibition results from a neuro-immune interaction involving a neuron-to-microglia-to-neuron signalling cascade.

References

  1. Agarwal N, Pacher P, Tegeder I, Amaya F, Constantin CE, Brenner GJ, Rubino T, Michalski CW, Marsicano G, Monory K, Mackie K, Marian C, Batkai S, Parolaro D, Fischer MJ, Reeh P, Kunos G, Kress M, Lutz B, Woolf CJ, Kuner R (2007) Cannabinoids mediate analgesia largely via peripheral type 1 cannabinoid receptors in nociceptors. Nat Neurosci 10:870–879CrossRefPubMedGoogle Scholar
  2. Aguado F, Carmona MA, Pozas E, Aguilo A, Martinez-Guijarro FJ, Alcantara S, Borrell V, Yuste R, Ibanez CF, Soriano E (2003) BDNF regulates spontaneous correlated activity at early developmental stages by increasing synaptogenesis and expression of the K+/Cl co-transporter KCC2. Development 130:1267–1280CrossRefPubMedGoogle Scholar
  3. Ahmadi S, Lippross S, Neuhuber WL, Zeilhofer HU (2002) PGE(2) selectively blocks inhibitory glycinergic neurotransmission onto rat superficial dorsal horn neurons. Nat Neurosci 5:34–40CrossRefPubMedGoogle Scholar
  4. Asiedu M, Ossipov MH, Kaila K, Price TJ (2009) Acetazolamide and midazolam act synergistically to inhibit neuropathic pain. PainGoogle Scholar
  5. Baba H, Ji RR, Kohno T, Moore KA, Ataka T, Wakai A, Okamoto M, Woolf CJ (2003) Removal of GABAergic inhibition facilitates polysynaptic A fiber-mediated excitatory transmission to the superficial spinal dorsal horn. Mol Cell Neurosci 24:818–830CrossRefPubMedGoogle Scholar
  6. Baba H, Kohno T, Okamoto M, Goldstein PA, Shimoji K, Yoshimura M (1998) Muscarinic facilitation of GABA release in substantia gelatinosa of the rat spinal dorsal horn. J Physiol 508 (Pt 1):83–93PubMedGoogle Scholar
  7. Baccei ML, Fitzgerald M (2004) Development of GABAergic and glycinergic transmission in the neonatal rat dorsal horn. J Neurosci 24:4749–4757CrossRefPubMedGoogle Scholar
  8. Bardoni R, Ghirri A, Salio C, Prandini M, Merighi A (2007) BDNF-mediated modulation of GABA and glycine release in dorsal horn lamina II from postnatal rats. Dev Neurobiol 67:960–975CrossRefPubMedGoogle Scholar
  9. Baufreton J, Atherton JF, Surmeier DJ, Bevan MD (2005) Enhancement of excitatory synaptic integration by GABAergic inhibition in the subthalamic nucleus. J Neurosci 25:8505–8517CrossRefPubMedGoogle Scholar
  10. Bevan MD, Magill PJ, Hallworth NE, Bolam JP, Wilson CJ (2002) Regulation of the timing and pattern of action potential generation in rat subthalamic neurons in vitro by GABA-A IPSPs. J Neurophysiol 87:1348–1362PubMedGoogle Scholar
  11. Blaesse P, Guillemin I, Schindler J, Schweizer M, Delpire E, Khiroug L, Friauf E, Nothwang HG (2006) Oligomerization of KCC2 correlates with development of inhibitory neurotransmission. J Neurosci 26:10407–10419CrossRefPubMedGoogle Scholar
  12. Castro-Lopes JM, Tavares I, Coimbra A (1993) GABA decreases in the spinal cord dorsal horn after peripheral neurectomy. Brain Res 620:287–291CrossRefPubMedGoogle Scholar
  13. Castro-Lopes JM, Tölle TR, Pan B, Zieglgänsberger W (1994) Expression of GAD mRNA in spinal cord neurons of normal and monoarthritic rats. Brain Res Mol Brain Res 26:169–176CrossRefPubMedGoogle Scholar
  14. Chéry N, De Koninck Y (2000) GABAB receptors are the first target of GABA released at lamina I inhibitory synapses in the rat spinal cord. J Neurophysiol 84:1006–1011PubMedGoogle Scholar
  15. Choi IS, Cho JH, Jeong SG, Hong JS, Kim SJ, Kim J, Lee MG, Choi BJ, Jang IS (2008) GABA(B) receptor-mediated presynaptic inhibition of glycinergic transmission onto substantia gelatinosa neurons in the rat spinal cord. Pain 138:330–342CrossRefPubMedGoogle Scholar
  16. Cooper EJ, Johnston GA, Edwards FA (1999) Effects of a naturally occurring neurosteroid on GABAA IPSCs during development in rat hippocampal or cerebellar slices. J Physiol 521 Pt 2:437–449CrossRefPubMedGoogle Scholar
  17. Cordero-Erausquin M, Coull JA, Boudreau D, Rolland M, De Koninck Y (2005) Differential maturation of GABA action and anion reversal potential in spinal lamina I neurons; impact of chloride extrusion capacity. J Neurosci 25:9613–9623CrossRefPubMedGoogle Scholar
  18. Cordero-Erausquin M, Pons S, Faure P, Changeux JP (2004) Nicotine differentially activates inhibitory and excitatory neurons in the dorsal spinal cord. Pain 109:308–318CrossRefPubMedGoogle Scholar
  19. Coull JA, Beggs S, Boudreau D, Boivin D, Tsuda M, Inoue K, Gravel C, Salter MW, De Koninck Y (2005) BDNF from microglia causes the shift in neuronal anion gradient underlying neuropathic pain. Nature 438:1017–1021CrossRefPubMedGoogle Scholar
  20. Coull JA, Boudreau D, Bachand K, Prescott SA, Nault F, Sik A, De Koninck P, De Koninck Y (2003) Trans-synaptic shift in anion gradient in spinal lamina I neurons as a mechanism of neuropathic pain. Nature 424:938–942CrossRefPubMedGoogle Scholar
  21. Cramer SW, Baggott C, Cain J, Tilghman J, Allcock B, Miranpuri G, Rajpal S, Sun D, Resnick D (2008) The role of cation-dependent chloride transporters in neuropathic pain following spinal cord injury. Mol Pain 4:36PubMedGoogle Scholar
  22. De Koninck Y (2007) Altered chloride homeostasis in neurological disorders: a new target. Curr Opin Pharmacol 7:93–99CrossRefPubMedGoogle Scholar
  23. Eaton MJ, Plunkett JA, Karmally S, Martinez MA, Montanez K (1998) Changes in GAD- and GABA- immunoreactivity in the spinal dorsal horn after peripheral nerve injury and promotion of recovery by lumbar transplant of immortalized serotonergic precursors. J Chem Neuroanat 16:57–72CrossRefPubMedGoogle Scholar
  24. Engelman HS, Anderson RL, Daniele C, Macdermott AB (2006) Presynaptic alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors modulate release of inhibitory amino acids in rat spinal cord dorsal horn. Neuroscience 139:539–553CrossRefPubMedGoogle Scholar
  25. Farquhar-Smith WP, Egertova M, Bradbury EJ, McMahon SB, Rice AS, Elphick MR (2000) Cannabinoid CB(1) receptor expression in rat spinal cord. Mol Cell Neurosci 15:510–521CrossRefPubMedGoogle Scholar
  26. Fukushima T, Ohtsubo T, Tsuda M, Yanagawa Y, Hori Y (2009) Facilitatory actions of serotonin type 3 receptors on GABAergic inhibitory synaptic transmission in the spinal superficial dorsal horn. J Neurophysiol 102:1459–1471CrossRefPubMedGoogle Scholar
  27. Gamba G (2005) Molecular physiology and pathophysiology of electroneutral cation-chloride cotransporters. Physiol Rev 85:423–493CrossRefPubMedGoogle Scholar
  28. Gassner M, Ruscheweyh R, Sandkuhler J (2009) Direct excitation of spinal GABAergic interneurons by noradrenaline. Pain 145:204–210CrossRefPubMedGoogle Scholar
  29. Guhring H, Gorig M, Ates M, Coste O, Zeilhofer HU, Pahl A, Rehse K, Brune K (2000) Suppressed injury-induced rise in spinal prostaglandin E2 production and reduced early thermal hyperalgesia in iNOS-deficient mice. J Neurosci 20:6714–6720PubMedGoogle Scholar
  30. Harrison NL, Vicini S, Barker JL (1987) A steroid anesthetic prolongs inhibitory postsynaptic currents in cultured rat hippocampal neurons. J Neurosci 7:604–609PubMedGoogle Scholar
  31. Harvey RJ, Depner UB, Wassle H, Ahmadi S, Heindl C, Reinold H, Smart TG, Harvey K, Schutz B, Abo-Salem OM, Zimmer A, Poisbeau P, Welzl H, Wolfer DP, Betz H, Zeilhofer HU, Muller U (2004) GlyR alpha3: an essential target for spinal PGE2-mediated inflammatory pain sensitization. Science 304:884–887CrossRefPubMedGoogle Scholar
  32. Hewitt SA, Wamsteeker JI, Kurz EU, Bains JS (2009) Altered chloride homeostasis removes synaptic inhibitory constraint of the stress axis. Nat Neurosci 12:438–443CrossRefPubMedGoogle Scholar
  33. Honda S, Sasaki Y, Ohsawa K, Imai Y, Nakamura Y, Inoue K, Kohsaka S (2001) Extracellular ATP or ADP induce chemotaxis of cultured microglia through Gi/o-coupled P2Y receptors. J Neurosci 21:1975–1982PubMedGoogle Scholar
  34. Hosl K, Reinold H, Harvey RJ, Muller U, Narumiya S, Zeilhofer HU (2006) Spinal prostaglandin E receptors of the EP2 subtype and the glycine receptor alpha3 subunit, which mediate central inflammatory hyperalgesia, do not contribute to pain after peripheral nerve injury or formalin injection. Pain 126:46–53CrossRefPubMedGoogle Scholar
  35. Hugel S, Schlichter R (2003) Convergent control of synaptic GABA release from rat dorsal horn neurones by adenosine and GABA autoreceptors. J Physiol 551:479–489CrossRefPubMedGoogle Scholar
  36. Ibuki T, Hama AT, Wang XT, Pappas GD, Sagen J (1997) Loss of GABA-immunoreactivity in the spinal dorsal horn of rats with peripheral nerve injury and promotion of recovery by adrenal medullary grafts. Neuroscience 76:845–858CrossRefPubMedGoogle Scholar
  37. Inoue K (2008) Purinergic systems in microglia. Cell Mol Life Sci 65:3074–3080CrossRefPubMedGoogle Scholar
  38. Jang IS, Rhee JS, Kubota H, Akaike N, Akaike N (2001) Developmental changes in P2X purinoceptors on glycinergic presynaptic nerve terminals projecting to rat substantia gelatinosa neurones. J Physiol 536:505–519CrossRefPubMedGoogle Scholar
  39. Jin X, Huguenard JR, Prince DA (2005) Impaired Cl extrusion in layer V pyramidal neurons of chronically injured epileptogenic neocortex. J Neurophysiol 93:2117–2126CrossRefPubMedGoogle Scholar
  40. Jolivalt CG, Lee CA, Ramos KM, Calcutt NA (2008) Allodynia and hyperalgesia in diabetic rats are mediated by GABA and depletion of spinal potassium-chloride co-transporters. Pain 140:48–57CrossRefPubMedGoogle Scholar
  41. Keller AF, Beggs S, Salter MW, De Koninck Y (2007) Transformation of the output of spinal lamina I neurons after nerve injury and microglia stimulation underlying neuropathic pain. Mol Pain 3:27CrossRefPubMedGoogle Scholar
  42. Keller AF, Breton JD, Schlichter R, Poisbeau P (2004) Production of 5alpha-reduced neurosteroids is developmentally regulated and shapes GABA(A) miniature IPSCs in lamina II of the spinal cord. J Neurosci 24:907–915CrossRefPubMedGoogle Scholar
  43. Kerchner GA, Wang GD, Qiu CS, Huettner JE, Zhuo M (2001) Direct presynaptic regulation of GABA/glycine release by kainate receptors in the dorsal horn: an ionotropic mechanism. Neuron 32:477–488CrossRefPubMedGoogle Scholar
  44. Khirug S, Huttu K, Ludwig A, Smirnov S, Voipio J, Rivera C, Kaila K, Khiroug L (2005) Distinct properties of functional KCC2 expression in immature mouse hippocampal neurons in culture and in acute slices. Eur J Neurosci 21:899–904CrossRefPubMedGoogle Scholar
  45. Khirug S, Yamada J, Afzalov R, Voipio J, Khiroug L, Kaila K (2008) GABAergic depolarization of the axon initial segment in cortical principal neurons is caused by the Na-K-2Cl cotransporter NKCC1. J Neurosci 28:4635–4639CrossRefPubMedGoogle Scholar
  46. Kiyosawa A, Katsurabayashi S, Akaike N, Pang ZP, Akaike N (2001) Nicotine facilitates glycine release in the rat spinal dorsal horn. J Physiol 536:101–110CrossRefPubMedGoogle Scholar
  47. Knabl J, Witschi R, Hosl K, Reinold H, Zeilhofer UB, Ahmadi S, Brockhaus J, Sergejeva M, Hess A, Brune K, Fritschy JM, Rudolph U, Mohler H, Zeilhofer HU (2008) Reversal of pathological pain through specific spinal GABAA receptor subtypes. Nature 451:330–334CrossRefPubMedGoogle Scholar
  48. Lu CR, Willcockson HH, Phend KD, Lucifora S, Darstein M, Valtschanoff JG, Rustioni A (2005) Ionotropic glutamate receptors are expressed in GABAergic terminals in the rat superficial dorsal horn. J Comp Neurol 486:169–178CrossRefPubMedGoogle Scholar
  49. Lu Y, Zheng J, Xiong L, Zimmermann M, Yang J (2008) Spinal cord injury-induced attenuation of GABAergic inhibition in spinal dorsal horn circuits is associated with down-regulation of the chloride transporter KCC2 in rat. J Physiol 586:5701–5715CrossRefPubMedGoogle Scholar
  50. Merighi A, Salio C, Ghirri A, Lossi L, Ferrini F, Betelli C, Bardoni R (2008) BDNF as a pain modulator. Prog Neurobiol 85:297–317CrossRefPubMedGoogle Scholar
  51. Miletic G, Miletic V (2008) Loose ligation of the sciatic nerve is associated with TrkB receptor-dependent decreases in KCC2 protein levels in the ipsilateral spinal dorsal horn. Pain 137:532–539CrossRefPubMedGoogle Scholar
  52. Mitchell SJ, Silver RA (2003) Shunting inhibition modulates neuronal gain during synaptic excitation. Neuron 38:433–445CrossRefPubMedGoogle Scholar
  53. Moore KA, Kohno T, Karchewski LA, Scholz J, Baba H, Woolf CJ (2002) Partial peripheral nerve injury promotes a selective loss of GABAergic inhibition in the superficial dorsal horn of the spinal cord. J Neurosci 22:6724–6731PubMedGoogle Scholar
  54. Palma E, Amici M, Sobrero F, Spinelli G, Di Angelantonio S, Ragozzino D, Mascia A, Scoppetta C, Esposito V, Miledi R, Eusebi F (2006) Anomalous levels of Cl transporters in the hippocampal subiculum from temporal lobe epilepsy patients make GABA excitatory. Proc Natl Acad Sci U S A 103:8465–8468CrossRefPubMedGoogle Scholar
  55. Payne JA, Rivera C, Voipio J, Kaila K (2003) Cation-chloride co-transporters in neuronal communication, development and trauma. Trends Neurosci 26:199–206CrossRefPubMedGoogle Scholar
  56. Pernia-Andrade AJ, Kato A, Witschi R, Nyilas R, Katona I, Freund TF, Watanabe M, Filitz J, Koppert W, Schuttler J, Ji G, Neugebauer V, Marsicano G, Lutz B, Vanegas H, Zeilhofer HU (2009) Spinal endocannabinoids and CB1 receptors mediate C-fiber-induced heterosynaptic pain sensitization. Science 325:760–764CrossRefPubMedGoogle Scholar
  57. Poisbeau P, Patte-Mensah C, Keller AF, Barrot M, Breton JD, Luis-Delgado OE, Freund-Mercier MJ, Mensah-Nyagan AG, Schlichter R (2005) Inflammatory pain upregulates spinal inhibition via endogenous neurosteroid production. J Neurosci 25:11768–11776CrossRefPubMedGoogle Scholar
  58. Polgar E, Gray S, Riddell JS, Todd AJ (2004) Lack of evidence for significant neuronal loss in laminae I-III of the spinal dorsal horn of the rat in the chronic constriction injury model. Pain 111:144–150CrossRefPubMedGoogle Scholar
  59. Polgar E, Hughes DI, Arham AZ, Todd AJ (2005) Loss of neurons from laminas I-III of the spinal dorsal horn is not required for development of tactile allodynia in the spared nerve injury model of neuropathic pain. J Neurosci 25:6658–6666CrossRefPubMedGoogle Scholar
  60. Prescott SA, Sejnowski TJ, De Koninck Y (2006) Reduction of anion reversal potential subverts the inhibitory control of firing rate in spinal lamina I neurons: a biophysical basis for neuropathic pain. Mol Pain 2:32CrossRefPubMedGoogle Scholar
  61. Price TJ, Cervero F, De Koninck Y (2005) Role of cation-chloride-cotransporters (CCC) in pain and hyperalgesia. Curr Top Med Chem 5:547–555CrossRefPubMedGoogle Scholar
  62. Rinehart J, Maksimova YD, Tanis JE, Stone KL, Hodson CA, Zhang J, Risinger M, Pan W, Wu D, Colangelo CM, Forbush B, Joiner CH, Gulcicek EE, Gallagher PG, Lifton RP (2009) Sites of regulated phosphorylation that control K-Cl cotransporter activity. Cell 138:525–536CrossRefPubMedGoogle Scholar
  63. Rivera C, Li H, Thomas-Crusells J, Lahtinen H, Viitanen T, Nanobashvili A, Kokaia Z, Airaksinen MS, Voipio J, Kaila K, Saarma M (2002) BDNF-induced TrkB activation down-regulates the K+-Cl cotransporter KCC2 and impairs neuronal Cl extrusion. J Cell Biol 159:747–752CrossRefPubMedGoogle Scholar
  64. Rivera C, Voipio J, Thomas-Crusells J, Li H, Emri Z, Sipila S, Payne JA, Minichiello L, Saarma M, Kaila K (2004) Mechanism of activity-dependent downregulation of the neuron-specific K-Cl cotransporter KCC2. J Neurosci 24:4683–4691CrossRefPubMedGoogle Scholar
  65. Samad TA, Moore KA, Sapirstein A, Billet S, Allchorne A, Poole S, Bonventre JV, Woolf CJ (2001) Interleukin-1beta-mediated induction of Cox-2 in the CNS contributes to inflammatory pain hypersensitivity. Nature 410:471–475CrossRefPubMedGoogle Scholar
  66. Scholz J, Broom DC, Youn DH, Mills CD, Kohno T, Suter MR, Moore KA, Decosterd I, Coggeshall RE, Woolf CJ (2005) Blocking caspase activity prevents transsynaptic neuronal apoptosis and the loss of inhibition in lamina II of the dorsal horn after peripheral nerve injury. J Neurosci 25:7317–7323CrossRefPubMedGoogle Scholar
  67. Sherman SE, Loomis CW (1994) Morphine insensitive allodynia is produced by intrathecal strychnine in the lightly anesthetized rat. Pain 56:17–29CrossRefPubMedGoogle Scholar
  68. Sherman SE, Luo L, Dostrovsky JO (1997) Spinal strychnine alters response properties of nociceptive-specific neurons in rat medial thalamus. J Neurophysiol 78:628–637PubMedGoogle Scholar
  69. Sivilotti L, Woolf CJ (1994) The contribution of GABAA and glycine receptors to central sensitization: disinhibition and touch-evoked allodynia in the spinal cord. J Neurophysiol 72:169–179PubMedGoogle Scholar
  70. Somers DL, Clemente FR (2002) Dorsal horn synaptosomal content of aspartate, glutamate, glycine and GABA are differentially altered following chronic constriction injury to the rat sciatic nerve. Neurosci Lett 323:171–174CrossRefPubMedGoogle Scholar
  71. Sorkin LS, Puig S (1996) Neuronal model of tactile allodynia produced by spinal strychnine: effects of excitatory amino acid receptor antagonists and a μ-opiate receptor agonist. Pain 68:283–292CrossRefPubMedGoogle Scholar
  72. Torsney C, Macdermott AB (2006) Disinhibition opens the gate to pathological pain signaling in superficial neurokinin 1 receptor-expressing neurons in rat spinal cord. J Neurosci 26:1833–1843CrossRefPubMedGoogle Scholar
  73. Trang T, Beggs S, Salter MW (2006) Purinoceptors in microglia and neuropathic pain. Pflugers Arch 452:645–652CrossRefPubMedGoogle Scholar
  74. Trang T, Beggs S, Wan X, Salter MW (2009) P2X4-receptor mediated synthesis and release of brain-derived neurotrophic factor in microglia is dependent on calcium and p38-mitogen-activated protein kinase activation. J Neurosci 29:3518–3528CrossRefPubMedGoogle Scholar
  75. Tsuda M, Shigemoto-Mogami Y, Koizumi S, Mizokoshi A, Kohsaka S, Salter MW, Inoue K (2003) P2X4 receptors induced in spinal microglia gate tactile allodynia after nerve injury. Nature 424:778–783CrossRefPubMedGoogle Scholar
  76. Ulmann L, Hatcher JP, Hughes JP, Chaumont S, Green PJ, Conquet F, Buell GN, Reeve AJ, Chessell IP, Rassendren F (2008) Up-regulation of P2X4 receptors in spinal microglia after peripheral nerve injury mediates BDNF release and neuropathic pain. J Neurosci 28:11263–11268CrossRefPubMedGoogle Scholar
  77. Vergnano AM, Salio C, Merighi A (2004) NK1 receptor activation leads to enhancement of inhibitory neurotransmission in spinal substantia gelatinosa neurons of mouse. Pain 112:37–47CrossRefPubMedGoogle Scholar
  78. Walker JM, Hohmann AG (2005) Cannabinoid mechanisms of pain suppression. Handb Exp Pharmacol 509–554Google Scholar
  79. Wang XL, Zhang HM, Li DP, Chen SR, Pan HL (2006) Dynamic regulation of glycinergic input to spinal dorsal horn neurones by muscarinic receptor subtypes in rats. J Physiol 571:403–413CrossRefPubMedGoogle Scholar
  80. Watanabe M, Wake H, Moorhouse AJ, Nabekura J (2009) Clustering of neuronal K+-Cl cotransporters in lipid rafts by tyrosine phosphorylation. J Biol Chem 284(41):27980–27988CrossRefPubMedGoogle Scholar
  81. Yaksh TL (1989) Behavioral and autonomic correlates of the tactile evoked allodynia produced by spinal glycine inhibition: effects of modulatory receptor systems and excitatory amino acid antagonists. Pain 37:111–123CrossRefPubMedGoogle Scholar
  82. Zeilhofer HU, Brune K (2006) Analgesic strategies beyond the inhibition of cyclooxygenases. Trends Pharmacol Sci 27:467–474CrossRefPubMedGoogle Scholar
  83. Zeilhofer HU, Mohler H, Di LA (2009) GABAergic analgesia: new insights from mutant mice and subtype-selective agonists. Trends Pharmacol Sci 30:397–402CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • Charalampos Labrakakis
    • 1
    • 2
  • Francesco Ferrini
  • Yves De Koninck
  1. 1.Division Cellular neurobiologyCentre de recherche université Laval Robert GiffardQuebecCanada
  2. 2.Department of Biological Applications and TechnologyUniversity of IoanninaIoanninaGreece

Personalised recommendations