In animal models of chronic pain, we have shown that persistent pain, but not acute pain, epigenetically suppresses the output activities of gad65 gene and consequently causes impaired inhibitory function of GABAergic synapses in central pain-modulating neurons, contributing to the development of persistent pain sensitization. These results are supported by observations in gad65–/– mice showing impaired GABA synaptic function in the same neurons and sensitized pain behavior. In addition, histone hyperacetylation overcomes these molecular and synaptic changes by promoting gad65 output activities, thereby relieving the sensitized behavior of persistent pain.
Chronic pain involves altered expression of many genes through unknown mechanisms35
. In drug addiction, histone H3 and H4 acetylation modulates the expression of several genes that regulate transcriptional activities, including Cdk5, c-fos, CREB
. In nerve injury-induced hypoesthesia, the C-fiber dysfunction is reportedly mediated by epigenetic upregulation of the transcriptional repressor neuron-restrictive silencer factor (NRSF), but the pain sensitization does not seem to involve NRSF upregulation17
. Interestingly, HDAC inhibitors reduce inflammatory pain by upregulating spinal metabotropic glutamate 2 receptors16
. The present study identifies gad65
as an important target gene of histone modifications induced by persistent pain conditions, providing a potential epigenetic mechanism for the development of chronic pain.
GAD65 is preferentially targeted to presynaptic terminals of central neurons for GABA synthesis of synaptic vesicles and is required for active GABA synaptic release23,24,36
. Impaired GABA release would cause loss of GABAergic inhibition, leading to neuronal hyper-activation. For instance, nerve injury induces a loss of GABA inhibition in spinal neurons and enhancing GABA synaptic inhibition is effective in relieving injury-induced pain37-39
. Our results from rats and GAD65-deficient mice suggest that persistent pain of inflammation and neuropathy induces down-regulation of GAD65 activities, causing impairment of GABA synaptic inhibition in NRM and increasing the excitability of presumably pain-facilitating neurons. This is in line with recent reports that gad65–/–
mice display sensitized pain behavior40
and viral delivery of gad65
gene produces orofacial analgesia41
. Given the multifaceted mechanisms of chronic pain, it is likely that other genes, in addition to gad65
, also are targets of chronic pain-induced chromatin remodeling. This likely accounts for our observation of increased global histone acetylation by CFA, indicating increased activities of other genes to be investigated. While the pain-induced changes in GAD65 activities and GABA synaptic function indicate a likely neuronal locus, further studies are necessary to verify the localization of neuronal nuclei for the pain-induced histone modification.
GAD67 is a GABA-synthesizing enzyme preferentially for cytoplasmic GABA and its tonic release from neurons23
. Our data demonstrate a major role of presynaptic GAD65 in the pain mechanism, but that does not preclude the role of cellular GAD67, particularly in pain-induced adaptive changes in neuronal excitability for sensitized pain behaviors. While our results do not indicate pain-induced epigenetic modulation of gad67
gene through histone acetylation, GAD67 could participate in the pain mechanisms by decreasing tonic inhibition among neurons through reduced expression (
) for cellular GABA, by reducing synaptic GABA through some presynaptic functions, and by compensatory changes in response to GAD65 deficiency. Detailed mechanisms for the GAD67 roles warrant further studies.
How functionally distinct populations of NRM neurons adapt to chronic pain conditions and mediate sensitized pain behaviors in chronic pain remains unclear. Under normal conditions, opioids produce analgesia partly by reducing basal GABA transmission in NRM neurons, thereby activating the descending pain-inhibition system29,42
. Consistently, NRM-applied GABAA
receptor antagonists induce antinociception whereas GABAA
receptor agonists produce pain sensitization43,44
. However, under chronic pain conditions, considerable adaptive changes may have occurred both in GABA input activities onto different classes of NRM neurons and in GABAA
receptor properties. Our results (
) indicate that the pain-induced impairment of GABA synaptic inputs may preferentially affect and consequently hyper-activate MOR-expressing neurons. Activation of this neuron class presumably facilitates spinal pain transmission29,30
, contributing to the sensitized pain behaviors. Pain-induced decrease in GABA neurotransmission has also been reported recently in amygdala neurons from a rat model of arthritic pain45
. This GABA impairment-induced pain sensitization is further supported by our behavioral results that enhancing GABA inhibition by activating NRM GABAA
receptors produces an antinociceptive effect (
). Detailed molecular and cellular adaptations in GABA and glutamate synapses under chronic pain conditions are subjects of ongoing research.
Proinflammatory cytokines, released into the peripheral and central circulation by immune cells and glia in response to tissue inflammation and trauma, cause augmented pain33,34
, but the underlying cellular and molecular mechanisms remain unclear, particularly under chronic pain states. Proinflammatory cytokines may contribute to the development of chronic pain by sustained release from their sources and by their sensitized signaling mechanisms in nociceptors and central neurons to augment pain responses after healing. Our observations of both the relatively acute hyperalgesic effect of IL-1β and the ineffectiveness of repeated IL-1β administration in NRM on GAD65 expression indicate that this proinflammatory cytokine at least in NRM is not significantly involved in the GAD65-mediated pain mechanisms for the prolonged pain behaviors induced by SNL and CFA.
A common clinical problem at present is the transition from analgesic-responsive acute pain to chronic pain, of which some types are poorly responsive to currently available analgesics and often lead to long-term neuropsychiatric disorders such as depression, stress and drug addiction10,12,46,47
. While multiple forms of neuronal plasticity have been identified for the pathogenesis of chronic pain13
, the mechanisms underlying this critical transition from acute pain to chronic pain remain poorly understood. Our findings of the chromatin modifications emerging only after pain development for days indicate that the epigenetic mechanism of gad65
modulation might underlie the persistent phase of these pain conditions and this, together with regulations of multiple sets of other gene activities, could be an important initial step in this transition that leads to the development of chronic pain and associated disorders. In this regard, drugs such as HDAC inhibitors that overcome the effects of persistent pain on the output activities of gad65
and other target genes may serve as a new promising class of analgesics48
, as they could collectively block the upstream cause of pain-induced cascade alterations that lead to multiple system malfunctions and clinical symptoms in chronic pain development.