GABAA RECEPTOR AS A NOVEL REGULATOR OF PERIPHERAL PAIN SENSITIVITY AND LOCAL NEURON-GLIA INTERACTION

The pathogenesis of neuropathic pain and its chronicization process are still not fully elucidated. For this reason, therapies currently ongoing in the clinic are aimed to treat mostly the symptoms, considering poorly the target mechanisms causing those pathologies. The approach that we choose to investigate new pharmacological targets for the therapy of chronic pain, was to firstly investigate the mechanisms of nociception in physiological condition, finding new molecular pathways responsible for nociception modulation. Secondly, we were aimed to study the contribution of these targets in the pathological condition. In particular, in this PhD thesis, I focused on the characterization of the GABAergic system, primarily the GABAA receptors (GABAAR), along peripheral nociceptors axons (forming C-fibers), evaluating their putative role in the modulation of pain peripheral conduction, during different physio-pathological conditions. GABAAR mediate fast synaptic inhibition in the dendrites, soma and axons of mature neurons that maintain low intracellular Cl- concentrations. In immature neurons as well as mature somatosensory neurons, the intracellular Cl- concentration is physiologically elevated and this gives rise to depolarizing GABAAR currents. We here report that, in somatosensory unmyelinated C-fibre axons, GABAAR mediates depolarizing currents and that the magnitude and time course of GABAAR responses are determined by NKCC1 activity. GABA (1 µM-1 mM) depolarizing responses were mediated specifically by axonal GABAAR, indeed they were mimicked by muscimol (>1 µM) and gaboxadol (THIP; >1 µM) and blocked by bicuculline (50 µM). Depolarizing axonal responses to GABA were completely absent in mice lacking β3 GABAAR subunit (β3,fl/fl), in either all sensory neurons (AdvillinCRE) or in nociceptor neurons expressing NaV1.8 (snsCRE). qRT-PCR analyses suggested that the most common GABAAR composition in somatosensory DRG neurons was a2, b3 and g2. To examine the physiological role of axonal GABAAR, C-fibres were subjected to a sustained frequency challenge (3 min at 2.5 Hz) and three main effects were found. First, the amplitude of axonal GABA responses was increased, and this effect was deemed secondary to an NKCC1 mediated shift in E Cl-. Second, GABAAR activation increased the axonal conduction velocity of C-fibres. Third, axonal GABAAR was activated by endogenous ligands. Our results indicate that C-fibres sustained firing increases NKCC1 activity, shifting ECl- toward more positive values. In this condition, constitutive GABAAR currents maintain nociceptor conductance during sustained firing. Established the capability of peripheral GABAergic currents to stabilize nociceptor conductance during sustained activity, we investigated the GABAergic modulation in pathological conditions characterized by hypersensitivity of nociceptors. In this regard, we studied in vivo model of inflammatory pain on snsCRE;b3-/- mice. The outcomes indicated that GABAAR activity increment mechanical allodynia and prevent the insurgence of hyperalgesia. In addition, GABAAR activity prolonged the recovery time, maintaining the hypersensitive phenotype for a longer period of time (up to 4 weeks). This finding corroborated the hypothesis that GABAergic transmission within peripheral fibers is able to stabilize the physiological conduction of pain, although it appeared dangerous in pathological condition, promoting nociceptor hypersensitivity and chronicization. We found that ALLO, a neuroactive steroid able to activate GABAAR and endogenously synthesized in PNS, induces the release of the growth factor BDNF from SCs, which is able to target trkB receptors on axons, in turn inducing PKCe upregulation and activation. PKCe is a typical protein kinase known to be involved in the process of pain chronicization. Overall, these set of data suggested that GABAAR is involved in a complex paracrine mechanism mediated by SCs, which activate the GABAAR and subsequently modulate its hyperactivity by the BDNF release. In conclusion, the results presented in this PhD thesis highlight the novel role of peripheral GABAAR in the modulation of nociceptor conduction in different physio-pathological conditions affecting the peripheral nervous system. Moreover, our findings stressed the role of local neuron-glia interaction in such mechanisms. GABAAR is able to dynamically stabilize nociceptor conduction of action potential during sustained activity, preventing excessive C-fiber slowing. On the other hand, GABAAR promotes pain hypersensitive state after neuronal inflammation and prolong the symptoms, likely leading to the pain chronicization. SCs play a fundamental role in the regulation of GABAergic signalling along C-fibers, although further studies are needed to unveil this process. in this direction might leads to the individuation of new pharmacological targets, exploiting endogenous pathways to obtain selective peripheral treatments. Hopefully, the complete comprehension of all the mechanisms would lay the basis for future identification of novel, possibly local, therapeutic strategies for the treatment of peripheral neuropathies and associated chronic pain.