Diabetic neuropathy is a common form of peripheral neuropathy, yet the mechanisms responsible for pain in this disease are poorly understood. Alterations in the expression and function of voltage-gated tetrodotoxinresistant (TTX-R) sodium channels have been implicated in animal models of neuropathic pain, including models of diabetic neuropathy. We investigated the expression and function of TTX-sensitive (TTX-S) and TTX-R sodium channels in dorsal root ganglion (DRG) neurons and the responses to thermal hyperalgesia and mechanical allodynia in streptozotocin-treated rats between 4 -8 weeks after onset of diabetes. Diabetic rats demonstrated a significant reduction in the threshold for escape from innocuous mechanical pressure (allodynia) and a reduction in the latency to withdrawal from a noxious thermal stimulus (hyperalgesia). Both TTX-S and TTX-R sodium currents increased significantly in small DRG neurons isolated from diabetic rats. The voltage-dependent activation and steady-state inactivation curves for these currents were shifted negatively. TTX-S currents induced by fast or slow voltage ramps increased markedly in neurons from diabetic rats. Immunoblots and immunofluorescence staining demonstrated significant increases in the expression of Na v 1.3 (TTX-S) and Na v 1.7 (TTX-S) and decreases in the expression of Na v 1.6 (TTX-S) and Na v 1.8 (TTX-R) in diabetic rats. The level of serine/threonine phosphorylation of Na v 1.6 and Na v 1.8 increased in response to diabetes. In addition, increased tyrosine phosphorylation of Na v 1.6 and Na v 1.7 was observed in DRGs from diabetic rats. These results suggest that both TTX-S and TTX-R sodium channels play important roles and that differential phosphorylation of sodium channels involving both serine/threonine and tyrosine sites contributes to painful diabetic neuropathy.Diabetes mellitus is one of the most common chronic medical problems, affecting over 100 million people world-wide (1). Diabetic patients frequently exhibit one or more types of stimulus-evoked pain, including increased responsiveness to noxious stimuli (hyperalgesia) as well as hyper-responsiveness to normally innocuous stimuli (allodynia). The underlying mechanisms of persistent pain in diabetic patients remain poorly understood. In animal models of diabetes, hyperalgesia to nonnoxious thermal stimulation as well as tactile allodynia have been observed (2-4). The streptozotocin (STZ) 1 -induced diabetic rat model demonstrates many of the abnormalities observed in humans (5). Treatment with insulin prevents development or reverses many of the abnormalities observed in early painful diabetic neuropathy (6, 7).In diabetic rats with hyperalgesia, dorsal root ganglion (DRG) neurons display increased frequency of action potential generation in response to sustained suprathreshold mechanical stimulation (3, 4, 8 -10) and increased spontaneous activity (11). Both effects are thought to contribute to the sensation of pain. Voltage-gated sodium channels generate and propagate action potentials in exc...

Voltage-gated sodium channel (Na v 1) ␤2 subunits modulate channel gating, assembly, and cell-surface expression in CNS neurons in vitro and in vivo. ␤2 expression increases in sensory neurons after nerve injury, and development of mechanical allodynia in the spared nerve injury model is attenuated in ␤2-null mice. Thus, we hypothesized that ␤2 modulates electrical excitability in dorsal root ganglion (DRG) neurons in vivo. We compared sodium currents (I Na ) in small DRG neurons from ␤2 ϩ/ϩ and ␤2 Ϫ/Ϫ mice to determine the effects of ␤2 on tetrodotoxin-sensitive (TTX-S) and tetrodotoxin-resistant (TTX-R) Na v 1 in vivo. Small-fast DRG neurons acutely isolated from ␤2 Ϫ/Ϫ mice showed significant decreases in TTX-S I Na compared with ␤2 ϩ/ϩ neurons. This decrease included a 51% reduction in maximal sodium conductance with no detectable changes in the voltage dependence of activation or inactivation. TTX-S, but not TTX-R, I Na activation and inactivation kinetics in these cells were slower in ␤2 Ϫ/Ϫ mice compared with controls. The selective regulation of TTX-S I Na was supported by reductions in transcript and protein levels of TTX-S Na v 1s, particularly Na v 1.7. Low-threshold mechanical sensitivity was preserved in ␤2 Ϫ/Ϫ mice, but they were more sensitive to noxious thermal stimuli than wild type whereas their response during the late phase of the formalin test was attenuated. Our results suggest that ␤2 modulates TTX-S Na v 1 mRNA and protein expression resulting in increased TTX-S I Na and increases the rates of TTX-S Na v 1 activation and inactivation in small-fast DRG neurons in vivo. TTX-R I Na were not significantly modulated by ␤2.

BACKGROUNDS & AIMS Chronic stress alters the hypothalamic–pituitary–adrenal axis, increases gut motility, and increases perception of visceral pain. We investigated whether epigenetic mechanisms regulate chronic stress-induced visceral pain in the peripheral nervous systems of rats. METHODS Male rats were subjected to 1 hr water-avoidance stress each day, or given daily subcutaneous injections of corticosterone, for 10 consecutive days. L4–L5 and L6–S2 dorsal root ganglia (DRG) were collected and compared between stressed and control rats (placed for 1 hour each day in a tank without water). Levels of cannabinoid receptor 1 (CNR1), DNA (cytosine-5-)-methyltransferase 1 (DNMT1), transient receptor potential vanilloid type 1 (TRPV1), and EP300 were knocked down in DRG neurons in situ with small interfering RNAs. We measured DNA methylation and histone acetylation at genes encoding the glucocorticoid receptor (NR3C1), CNR1, and TRPV1. Visceral pain was measured in response to colorectal distention. RESULTS Chronic stress was associated with increased methylation of the Nr3c1 promoter and reduced expression of this gene in L6–S2, but not L4–L5, DRGs. Stress was also associated with upregulation in DNMT1-associated methylation of the Cnr1 promoter and downregulation of glucocorticoid receptor-mediated expression of CNR1 in L6–S2, but not L4–L5, DRGs. Concurrently, chronic stress increased expression of the histone acetyltransferase EP300 and increased histone acetylation at the Trpv1 promoter and expression of the TRPV1 receptor in L6–S2 DRG neurons. Knockdown of DNMT1 and EP300 in L6–S2 DRG neurons of rats reduced DNA methylation and histone acetylation, respectively, and prevented chronic stress-induced increases in visceral pain. CONCLUSIONS Chronic stress increases DNA methylation and histone acetylation of genes that regulate visceral pain sensation in the peripheral nervous system of rats. Blocking epigenetic regulatory pathways in specific regions of the spinal cord might be developed to treat patients with chronic abdominal pain.