This is a complex study of the intricate cellular and molecular mechanisms for the
maintenance of chronic pain of different aetiology in the dorsal horn (DH) of the
spinal cord. By employing different techniques and varied approaches, this study
demonstrates the impaired trafficking of AMPA receptors in sensory neurons of the
DH in persistent pain conditions. This includes internalization of GluR2-containing
AMPA receptors from the synapses between primary afferents and DH neurons,
whereas insertion of GluR1-containing, Ca2+-permeable AMPA receptors into the
extrasynaptic plasma membranes of sensory neurons. The molecular mechanism of
altered AMPA receptor trafficking in the DH neurons has been deciphered, showing
the involvement of numerous intracellular proteins (stargazin, ABP/GRIP, PICK1),
with the key role of РKС subtype α, a ubiquitous intracellular enzyme, in regulation
of GluR1 and GluR2 membrane translocations. The obtained data provide evidence
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for the causally linked changes between AMPA receptor trafficking and the balanced
synaptic excitation and inhibition within the DH neuronal networks, leading to the
hyperexcitability of the DH – the phenomenon of central sensitization. The latter is
thought to represent the mechanistic basis behind chronic pain of different origins. In
this study, it has been demonstrated, for the first time, that altered AMPA receptor
trafficking occurs in a cell-type-specific manner, shifting the balance between
synaptic excitation and inhibition differently for subpopulations of DH neurons. The
revealed causality between the altered AMPA receptors at the cellular level and the
chronic pain maintenance in vivo provides evidence for the key role of spinal
mechanisms in chronification of the emerged pain. Hence it ensures that developing
strategies for targeting the central mechanisms (rather than peripheral ones) would
succeed in the cure of chronic pain. With this, the next studies were, therefore,
focusing on elaborating new approaches to the treatment of chronic pain via targeting
the established molecular mechanism of AMPA receptor trafficking in the DH
neurons. First, an experimental methodology was evolved to deliver compounds of
interests site-specifically, using surgically implanted catheters for the local,
intrathecal drug delivery to the lumbar spinal cord segments, prone to minimize
potential adverse effects. The therapeutic efficacy of targeting the impaired spinal
AMPA receptors was next tested by using a pharmacological approach, i.e. selective
inhibition of Ca2+-permeable AMPA receptors. For this, it has been implemented a
class of novel high potent blockers of Ca2+-permeable AMPA receptors, the openchannel
antagonists acting in an activity-dependent manner (dicationic compounds
IEM-1460 and IEM-1925). The obtained data confirmed the antinociceptive effects of
the two compounds tested, with no side effects detectable upon activity-dependent
inhibition of spinal Ca2+-permeable AMPA receptors. The next strategy consisted of
targeting spinal РKСα – as a prerequisite for the impaired AMPA receptor trafficking
– via either pharmacological or genetic inhibition. A novel selective antagonist of
РKСα, C2-4 peptide, was probed, which first revealed the antinociceptive effect here.
For gene-therapy, the gene-silencing approach has been utilized to knock-down spinal
РKСα with antisense oligonucleotides delivered to the lumbar spinal cord in an
experimental model of persistent inflammatory pain. The therapeutic outcomes of
gene-therapy included substantial relief in inflammatory pain (alleviated nociceptive
hypersensitivity of different modalities) and shortening of the pain maintenance, in
either pre- or post-treatment. This pain relief was paralleled by a decline in the
impaired AMPA receptor trafficking at the cellular level, as confirmed by
electrophysiological recordings made from the DH neurons from animals with
persistent peripheral inflammation. Together, these data provide fundamental
knowledge for the dynamic AMPA receptor trafficking in sensory neurons and open a
new line of studies to focus on the mechanism-based treatment of chronic pain
through interference with molecular mechanisms for nociceptive signalling in central
pain pathways.
