Silencing the α2 subunit of γ-aminobutyric acid type A receptors in rat dorsal root ganglia reveals its major role in antinociception posttraumatic nerve injury.

BACKGROUND: Neuropathic pain (NPP) is likely the result of repetitive high-frequency bursts of peripheral afferent activity leading to long-lasting changes in synaptic plasticity in the spinal dorsal horn. Drugs that promote γ-aminobutyric acid (GABA) activity in the dorsal horn provide partial relief of neuropathic symptoms. The authors examined how in vivo silencing of the GABA receptor type A (GABAA) α2 gene in dorsal root ganglia (DRG) controls NPP. METHODS: After crush injury to the right sciatic nerve of female rats, the α2 GABAA antisense and mismatch oligodeoxynucleotides or NO-711 (a GABA uptake inhibitor) were applied to the L5 DRG. In vivo behavioral assessment of nociception was conducted before the injury and ensuing 10 days (n = 4 to 10). In vitro quantification of α2 GABAA protein and electrophysiological studies of GABAA currents were performed on acutely dissociated L5 DRG neurons at relevant time points (n = 6 to 14). RESULTS: NPP postcrush injury of a sciatic nerve in adult female rats coincides with significant down-regulation of the α2 subunit expression in the ipsilateral DRG (approximately 30%). Selective down-regulation of α2 expression in DRGs significantly worsens mechanical (2.55 ± 0.75 to 5.16 ± 1.16) and thermal (7.97 ± 0.96 to 5.51 ± 0.75) hypersensitivity in crush-injured animals and causes development of significant mechanical (2.33 ± 0.40 to 5.00 ± 0.33) and thermal (10.80 ± 0.29 to 7.34 ± 0.81) hypersensitivity in sham animals (data shown as mean ± SD). Conversely, up-regulation of endogenous GABA via blockade of its uptake in DRG alleviates NPP. CONCLUSION: The GABAA receptor in the DRG plays an important role in pathophysiology of NPP caused by sciatic nerve injury and represents promising target for novel pain therapies.

Cell-specific alterations of T-type calcium current in painful diabetic neuropathy enhance excitability of sensory neurons.

Recent data indicate that T-type Ca2+ channels are amplifiers of peripheral pain signals, but their involvement in disorders of sensory neurons such as those associated with diabetes is poorly understood. To address this issue, we used a combination of behavioral, immunohistological, molecular, and electrophysiological studies in rats with streptozotocin (N-[methylnitrosocarbamoil]-D-glucosamine)-induced early diabetic neuropathy. We found that, in parallel with the development of diabetes-induced pain, T-type current density increased by twofold in medium-size cells from L4-L5 dorsal root ganglia (DRG) with a depolarizing shift in steady-state inactivation. This not only correlated closely with more prominent afterdepolarizing potentials (ADPs) but also increased cellular excitability manifested as a lower threshold for burst firing in diabetic than in control cells. T-type currents and ADPs were potently inhibited by nickel and enhanced by L-cysteine, suggesting that the Ca(V)3.2 T-type channel isoform was upregulated. Both control and diabetic DRG cells with ADPs stained positively for isolectin B4, but only diabetic cells responded robustly to capsaicin, suggesting enhanced nociceptive function. Because increased excitability of sensory neurons may result in such pathological perceptions of pain as hyperalgesia and allodynia, upregulation of T-type Ca2+ currents and enhanced Ca2+ entry into these cells could contribute to the development of symptoms in diabetic neuropathy.

New evidence that both T-type calcium channels and GABAA channels are responsible for the potent peripheral analgesic effects of 5alpha-reduced neuroactive steroids.

Neurosteroids are potent blockers of neuronal low-voltage activated (T-type) Ca(2+) channels and potentiators of GABA(A) ligand-gated channels, but their effects in peripheral pain pathways have not been studied previously. To investigate potential analgesic effects and the ion channels involved, we tested the ability of locally injected 5alpha-reduced neurosteroids to modulate peripheral thermal nociception to radiant heat in adult rats in vivo and to modulate GABA(A) and T-type Ca(2+) channels in vitro. The steroid anesthetic alphaxalone (ALPX), the endogenous neurosteroid allopregnanolone (3alpha5alphaP), and a related compound ((3alpha,5alpha,17beta)-3-hydroxyandrostane-17-carbonitrile, (ACN)), induced potent, dose-dependent, enantioselective anti-nociception in vivo and modulation of both T-type Ca(2+) currents and GABA(A)-mediated currents in vitro. Analgesic effects of ALPX were incompletely antagonized by co-injections of the GABA(A) receptor antagonist bicuculline. The neurosteroid analogue ((3alpha,5alpha)-3-hydroxy-13,24-cyclo-18,21-dinorchol-22-en-24-ol (CDNC24), a compound with GABAergic but not T-type activity, was not analgesic. However, (3beta,5alpha,17beta)-17-hydroxyestrane-3-carbonitrile (ECN)), which has effects on T-type channels but not on GABA(A) receptors, also induced potent enantioselective peripheral anti-nociception. ECN increased pain thresholds less than ALPX, 3alpha5alphaP and ACN. However, when an ineffective dose of CDNC24 was combined with ECN, anti-nociceptive activity was greatly enhanced, and this effect was bicuculline-sensitive. These results strongly suggest that GABA(A) channels do not contribute to baseline pain transmission, but they can enhance anti-nociception mediated by blockade of T-type Ca(2+) channels. In conclusion, we demonstrate that potent peripheral analgesia induced by 5alpha-reduced neurosteroid is mediated in part by effects on T-type Ca(2+) channels. Our results also reveal a role of GABA-gated ion channels in peripheral nociceptive signaling.

Mechanical and thermal anti-nociception in rats after systemic administration of verapamil.

Voltage-gated Ca(2+) channels expressed in neurons may contribute to nociceptive information processing. However, the role of L-type Ca(2+) channels in pain transmission is not well understood. In this study, we examined the effects of systemically administered verapamil, an antihypertensive agent and L-type Ca(2+) channel blocker, on mechanical and thermal withdrawal thresholds in rats. Intraperitoneal injections of verapamil induced dose-dependent (3-18 mg/kg) mechanical and thermal anti-nociception in adult rats without altering their sensorimotor abilities. Our data suggest that L-type Ca(2+) channels contribute to acute nociceptive signaling and that anti-nociceptive effects may result from the blockade of these channels.

Selective T-type calcium channel blockade alleviates hyperalgesia in ob/ob mice.

OBJECTIVE: Morbid obesity may be accompanied by diabetes and painful diabetic neuropathy, a poorly understood condition that is manifested by mechanical or thermal allodynia and hyperalgesia. Recent studies have highlighted the importance of T-type calcium channels (T-channels) in peripheral nociception; therefore, our goal was to examine the function of these channels in the pathophysiology and development of painful diabetic neuropathy. RESEARCH DESIGN AND METHODS: In vivo testing of mechanical and thermal sensation, morphometric peripheral nerve studies, and electrophysiological and biochemical measurements were used to characterize the role of T-channels and the development of painful diabetic neuropathy in leptin-deficient (ob/ob) mice. RESULTS: We found that ob/ob mice developed significant mechanical and thermal hypersensitivity early in life that coincided with hyperglycemia and was readily reversed with insulin therapy. These disturbances were accompanied by significant biophysical and biochemical modulation of T-channels in dorsal root ganglion neurons as measured by a large increase in the amplitude of T-currents and the expression of mRNA. The most prevalent subtype, alpha1H (Ca(v)3.2), was most strongly affected. Moreover, (3beta,5alpha,17beta)-17-hydroxyestrane-3-carbonitrile (ECN), a novel neuroactive steroid and selective T-channel antagonist, provided dose-dependent alleviation of neuropathic thermal and mechanical hypersensitivity in diabetic ob/ob mice. CONCLUSIONS: Our results indicate that pharmacological antagonism of T-channels is potentially an important novel therapeutic approach for the management of painful diabetic neuropathy.

Upregulation of the T-type calcium current in small rat sensory neurons after chronic constrictive injury of the sciatic nerve.

Recent data indicate that peripheral T-type Ca2+ channels are instrumental in supporting acute pain transmission. However, the function of these channels in chronic pain processing is less clear. To address this issue, we studied the expression of T-type Ca2+ currents in small nociceptive dorsal root ganglion (DRG) cells from L4-5 spinal ganglia of adult rats with neuropathic pain due to chronic constrictive injury (CCI) of the sciatic nerve. In control rats, whole cell recordings revealed that T-type currents, measured in 10 mM Ba2+ as a charge carrier, were present in moderate density (20 +/- 2 pA/pF). In rats with CCI, T-type current density (30 +/- 3 pA/pF) was significantly increased, but voltage- and time-dependent activation and inactivation kinetics were not significantly different from those in controls. CCI-induced neuropathy did not significantly change the pharmacological sensitivity of T-type current in these cells to nickel. Collectively, our results indicate that CCI-induced neuropathy significantly increases T-type current expression in small DRG neurons. Our finding that T-type currents are upregulated in a CCI model of peripheral neuropathy and earlier pharmacological and molecular studies suggest that T-type channels may be potentially useful therapeutic targets for the treatment of neuropathic pain associated with partial mechanical injury to the sciatic nerve.

Differential effects of endogenous cysteine analogs on peripheral thermal nociception in intact rats.

Previous in vivo studies indicate that locally injected redox-modulating agents can sensitize polymodal peripheral skin nociceptors resulting in acute changes in pain perception. Since endogenous thiol-modifying redox agents are normally present in the interstitial tissue, and could be found in higher concentration in certain conditions (e.g., tissue injury, inflammation, and ischemia), we designed this study to evaluate the peripheral nociceptive effects of locally injected endogenous-reducing cysteine analogs, L-cysteine, D-cysteine and D,L-homocysteine and endogenous-oxidizing cysteine analogs, L-cystine, D-cystine and D,L-homocystine using the acute model of thermal peripheral nociception in intact rats. We found that the reducing cysteine analogs induced potent dose- and time-dependent hyperalgesia and conversely the oxidizing cysteine analogs induced potent dose- and time-dependent analgesia. In the presence of 3betaOH, a novel neuroactive steroid and potent voltage-dependent blocker of T-type Ca2+ channels, the hyperalgesic effects of the reducing agents were diminished, whereas the analgesic effects of the oxidizing agents were enhanced strongly suggesting that the observed nociceptive effects were, at least in part, mediated via the peripheral T channels. Our findings imply that changes in the redox states of the peripheral nociceptors (favoring either reduced or oxidized forms of cysteine molecules) may function as a local intrinsic mechanism in controlling peripheral pain perception.

5beta-reduced neuroactive steroids are novel voltage-dependent blockers of T-type Ca2+ channels in rat sensory neurons in vitro and potent peripheral analgesics in vivo.

T-type Ca(2+) channels are believed to play an important role in pain perception, and anesthetic steroids such as alphaxalone and allopregnanolone, which have a 5alpha-configuration at the steroid A, B ring fusion, are known to inhibit T-type Ca(2+) channels and cause analgesia in a thermal nociceptive model (Soc Neurosci Abstr 29:657.9, 2003). To define further the structure-activity relationships for steroid analgesia, we synthesized and examined a series of 5beta-reduced steroids for their ability to induce thermal antinociception in rats when injected locally into the peripheral receptive fields of the nociceptors and studied their effects on T-type Ca(2+) channel function in vitro. We found that most of the steroids completely blocked T-type Ca(2+) currents in vitro with IC(50) values at a holding potential of -90 mV ranging from 2.8 to 40 microM. T current blockade exhibited mild voltage-dependence, suggesting that 5beta-reduced neuroactive steroids stabilize inactive states of the channel. For the most potent steroids, we found that other voltage-gated currents were not significantly affected at concentrations that produce nearly maximal blockade of T currents. All tested compounds induced dose-dependent analgesia in thermal nociceptive testing; the most potent effect (ED(50), 30 ng/100 microl) obtained with a compound [(3beta,5beta,17beta)-3-hydroxyandrostane-17-carbonitrile] that was also the most effective blocker of T currents. Compared with previously studied 5alpha-reduced steroids, these 5beta-reduced steroids are more efficacious blockers of neuronal T-type Ca(2+) channels and are potentially useful as new experimental reagents for understanding the role of neuronal T-type Ca(2+) channels in peripheral pain pathways.