San-Jue Hu

An experimental model for chronic compression of dorsal root ganglion produced by intervertebral foramen stenosis in the rat.

&NA; Under anesthesia and sterile surgery, a small stainless steel rod (4 mm in length and 0.5–0.8 mm in diameter) was inserted into the L5 intervertebral foramen in the rat, developing intervertebral foramen stenosis and hence producing a chronic steady compression of the dorsal root ganglion (DRG). The hind paw on the injured side exhibited a significant reduction in the latency of foot withdrawal to noxious heat and manifested a persistent heat hyperalgesia 5–35 days after surgery. Injection of 1% carrageenan into the intervertebral foramen, presumably causing inflammation of the DRG, also produced hyperalgesia to heat on the hind paw of the injured side 5–21 days after surgery. Extracellular electrophysiological recordings from myelinated dorsal root fibers were performed in vivo. Spontaneous activity was present in 21.5% of the fibers recorded from DRG neurons injured with chronic compression in contrast to 1.98% from uninjured DRG neurons. The pattern of spontaneous activity was periodic and bursting in 75.3% of the spontaneously active fibers. These neurons had a greatly enhanced sensitivity to mechanical stimulation of the injured DRG and a prolonged after discharge. In response to TEA, topically applied to the DRG, excitatory responses were evoked in the injured, but not the uninjured, DRG neurons. Application of this experimental model may further our understanding of the neural mechanisms by which chronic compression of DRG induces low back pain and sciatica.

Somata of nerve-injured sensory neurons exhibit enhanced responses to inflammatory mediators.

The effects of inflammatory mediators in modulating the activity of nerve‐injured dorsal root ganglion (DRG) neurons were studied in rats in an in vitro nerve‐DRG preparation 2–4 weeks after a loose ligation of the sciatic nerve (chronic constriction injury, CCI). An inflammatory soup (IS) of bradykinin, serotonin, prostaglandin E2 and histamine (each 10−5 M, pH=7.4) was applied topically to the DRG. Evoked responses were recorded extracellularly from teased dorsal root fibers or intracellularly with sharp electrodes from somata of DRG neurons with myelinated (A&bgr; and A&dgr;) or unmyelinated (C) axons. IS increased the rate of ongoing spontaneous activity recorded from dorsal root fibers of CCI neurons and evoked activity in a subpopulation of previously ‘silent’ fibers in CCI rats but not those of unoperated controls. In comparison with DRG somata of control rats, those of CCI become more excitable as evidenced by a lower threshold to depolarizing current and a greater depolarization in response to IS. Inflammatory mediators, by increasing the excitability of DRG neurons, may contribute to paresthesiae, pain and hyperalgesia after peripheral nerve injury.

Gabapentin selectively reduces persistent sodium current in injured type-A dorsal root ganglion neurons.

ABSTRACT It has been confirmed that the voltage‐gated persistent sodium currents mediate the generation of subthreshold membrane potential oscillations (SMPOs) and contribute to shaping repetitive firing. Our previous study indicated that gabapentin (GBP) administration induced a dose‐dependent inhibition of SMPO in chronically compressed dorsal root ganglion (CCD) neurons. To investigate the mechanisms and possible site(s) of action of GBP, the persistent sodium currents (INaP) were measured and the effects of GBP on INaP were examined in CCD neurons electrophysiologically in vitro. DRG neurons possess slow TTX‐sensitive inactivating sodium currents that significantly contribute to the generation of membrane oscillations by amplifying the resonance behavior. GBP reduced the resonant amplitude of DRG neurons as well as inhibiting the firing and SMPO induced by injection current, which was strongly due to the inhibitory effect on persistent sodium currents. Furthermore, we found that GBP (1–20 μM) administration inhibited the persistent sodium currents in dose‐dependent manner, while the changes of K+ and Ca2+ current minimally contributed to the effect of GBP on oscillation and resonant behavior of DRG neurons. In contrast, the amplitude and voltage‐dependence of transient sodium current were unchanged by GBP. The results suggest that GBP decreased the amplitude of resonance and abolished the SMPO of A‐type DRG neurons through the inhibition of INaP, and thus inhibited the SMPO dependent repetitive and bursting firings.

Blockade of persistent sodium currents contributes to the riluzole-induced inhibition of spontaneous activity and oscillations in injured DRG neurons.

In addition to a fast activating and immediately inactivating inward sodium current, many types of excitable cells possess a noninactivating or slowly inactivating component: the persistent sodium current (INaP). The INaP is found in normal primary sensory neurons where it is mediated by tetrodotoxin-sensitive sodium channels. The dorsal root ganglion (DRG) is the gateway for ectopic impulses that originate in pathological pain signals from the periphery. However, the role of INaP in DRG neurons remains unclear, particularly in neuropathic pain states. Using in vivo recordings from single medium- and large-diameter fibers isolated from the compressed DRG in Sprague-Dawley rats, we show that local application of riluzole, which blocks the INaP, also inhibits the spontaneous activity of A-type DRG neurons in a dose-dependent manner. Significantly, riluzole also abolished subthreshold membrane potential oscillations (SMPOs), although DRG neurons still responded to intracellular current injection with a single full-sized spike. In addition, the INaP was enhanced in medium- and large-sized neurons of the compressed DRG, while bath-applied riluzole significantly inhibited the INaP without affecting the transient sodium current (INaT). Taken together, these results demonstrate for the first time that the INaP blocker riluzole selectively inhibits INaP and thereby blocks SMPOs and the ectopic spontaneous activity of injured A-type DRG neurons. This suggests that the INaP of DRG neurons is a potential target for treating neuropathic pain at the peripheral level.

Theta-frequency membrane resonance and its ionic mechanisms in rat subicular pyramidal neurons.

The neuron population of the hippocampal formation exhibits oscillatory activity within the theta (theta) frequency band (4-10 Hz), and the intrinsic resonance properties of individual hippocampal neurons contribute to this network oscillation. The subiculum is the pivotal output region of the hippocampal formation and it is involved in many of the physiological and pathological functions of the limbic system. To study the characteristics and underlying mechanisms of resonance activity in subicular pyramidal neurons, we performed whole-cell patch-clamp recordings from these neurons in rat horizontal brain slices. We applied sinusoidal currents with constant amplitudes and linearly increasing frequencies to measure the resonance frequency of subicular pyramidal neurons. We found that the resonance frequency of subicular pyramidal neurons was about 2 Hz at room temperature and 4-6 Hz at 32-35 degrees C. The resonance frequency increased at hyperpolarized membrane potentials and decreased at depolarized membrane potentials. We also investigated three sub-threshold currents involved in the resonance: a slow hyperpolarization-activated cation current; an instantaneously activating, inwardly rectifying potassium current; and an inwardly persistent sodium current. The application of ZD7288 abolished the resonance hump, indicating that hyperpolarization-activated cation current generated resonance. The application of Ba(2+) enlarged the resonance hump at hyperpolarized potentials below -80 mV, indicating that inwardly rectifying potassium current attenuated resonance. The application of TTX suppressed the resonance at depolarized potentials, indicating that persistent sodium current amplified resonance when neurons were depolarized. Thus, there is a theta-frequency resonance mediated by hyperpolarization-activated cation current in subicular pyramidal neurons. This theta-frequency resonance of individual subicular pyramidal neurons may participate in the populations theta oscillation and contribute to the functions of the subiculum.

Gastrodin inhibits allodynia and hyperalgesia in painful diabetic neuropathy rats by decreasing excitability of nociceptive primary sensory neurons

Painful diabetic neuropathy (PDN) is a common complication of diabetes mellitus and adversely affects the patients’ quality of life. Evidence has accumulated that PDN is associated with hyperexcitability of peripheral nociceptive primary sensory neurons. However, the precise cellular mechanism underlying PDN remains elusive. This may result in the lacking of effective therapies for the treatment of PDN. The phenolic glucoside, gastrodin, which is a main constituent of the Chinese herbal medicine Gastrodia elata Blume, has been widely used as an anticonvulsant, sedative, and analgesic since ancient times. However, the cellular mechanisms underlying its analgesic actions are not well understood. By utilizing a combination of behavioral surveys and electrophysiological recordings, the present study investigated the role of gastrodin in an experimental rat model of STZ-induced PDN and to further explore the underlying cellular mechanisms. Intraperitoneal administration of gastrodin effectively attenuated both the mechanical allodynia and thermal hyperalgesia induced by STZ injection. Whole-cell patch clamp recordings were obtained from nociceptive, capsaicin-sensitive small diameter neurons of the intact dorsal root ganglion (DRG). Recordings from diabetic rats revealed that the abnormal hyperexcitability of neurons was greatly abolished by application of GAS. To determine which currents were involved in the antinociceptive action of gastrodin, we examined the effects of gastrodin on transient sodium currents (I NaT) and potassium currents in diabetic small DRG neurons. Diabetes caused a prominent enhancement of I NaT and a decrease of potassium currents, especially slowly inactivating potassium currents (I AS); these effects were completely reversed by GAS in a dose-dependent manner. Furthermore, changes in activation and inactivation kinetics of I NaT and total potassium current as well as I AS currents induced by STZ were normalized by GAS. This study provides a clear cellular basis for the peripheral analgesic action of gastrodin for the treatment of chronic pain, including PDN.

Evoked bursting in injured Aβ dorsal root ganglion neurons: A mechanism underlying tactile allodynia

Summary The evoked bursting discharges of the Aβ dorsal root ganglion neurons may contribute to the development of allodynia after nerve injury. Abstract Chronic compression of rat dorsal root ganglion (CCD) produced tactile allodynia accompanied with hyperexcitability of the myelinated Aβ dorsal root ganglion (DRG) neurons. The Aβ DRG neuron hyperexcitability exhibits as bursting discharges in response to peripherally evoked action potentials (evoked bursting [EB]). The incidence of EB was significantly increased after chronic compression of DRG (CCD) (43.3%) vs control (13.3%). EB was maintained by oscillation of the membrane potential, and its duration was increased when the membrane potential was depolarized. EB was found to coexist in some neurons with spontaneous bursting (SB), but EB always occurred at a more negative membrane potential than SB. Afterdischarges of the wide dynamic range neurons of the dorsal horn in the spinal cord in response to electrical stimulation of Aβ afferent nerve fibers were suppressed by blocking EB of the DRG neurons. CCD neurons with EB exhibited increased current density of persistent sodium current (INap) and hyperpolarization‐activated cation current (Ih) and decreased α‐dendrotoxin (α‐DTX) sensitive current (IDTX). The increased Ih activated by afterhyperpolarization of peripheral afferent action potential was necessary for EB generation and a balance between IDTX and INap might be necessary for EB maintenance. This study may suggest a role of EB of myelinated DRG neurons in development of allodynia after nerve injury and a potential pharmaceutical therapy in treating neuropathic allodynia.

Subthreshold membrane potential oscillation mediates the excitatory effect of norepinephrine in chronically compressed dorsal root ganglion neurons in the rat

Injured dorsal root ganglion (DRG) neurons often develop adrenergic sensitivity. To investigate the mechanisms of this phenomenon, the effects of norepinephrine (NE) on membrane potential of large‐ and medium‐sized A‐type neurons from chronically compressed DRG were recorded electrophysiologically in vitro. NE induced a depolarization in both control (26/36) and injured (56/62) neurons, whereas the incidence and amplitude of NE‐induced depolarization in the injured neurons were significantly higher than that in controls. Following NE‐induced depolarization, a subthreshold membrane potential oscillation (SMPO) was triggered or enhanced that initiated or increased repetitive firing in a fraction of injured neurons (15/56). After the SMPO was selectively abolished by application of tetrodotoxin (TTX), NE‐induced depolarization failed to produce repetitive firing, even with a greater depolarization. Application of Rp‐cAMPS (500 &mgr;M), a selective inhibitor of protein kinase A (PKA), decreased both SMPO and repetitive firing evoked by NE application or by intracellular current injection. Conversely, Sp‐cAMPS (500 &mgr;M), a PKA activator, had a facilitating effect on both the SMPO and the repetitive firing. These results strongly suggest that a PKA mediated triggering and enhancement of SMPO may be responsible for the excitatory effects of NE on sensory neurons in neuropathic rats.

Effects of gabapentin on spontaneous discharges and subthreshold membrane potential oscillation of type A neurons in injured DRG

&NA; Ectopic spontaneous discharges play a critical role for both initiation and maintenance of the neuropathic pain state. Gabapentin (GBP) has been shown to be effective in animal models of neuropathic pain as well as in chronic pain patients. To investigate the peripheral mechanisms of GBP, the effects of GBP on spontaneous discharges and subthreshold membrane potential oscillation (SMPO) of chronically compressed dorsal root ganglion (DRG) were examined electrophysiolocally in vitro. The rate of spontaneous discharges was transitorily enhanced when GBP was applied to the DRG. When the concentration was under 5 μM, only enhanced effect was observed, while spontaneous discharges were completely suppressed when the concentration of GBP was beyond 5 μM. The similar doses of GBP blocking the spontaneous discharges failed to block the propagation of impulses by electrical nerve stimulation. Furthermore, we found that the SMPO of injured DRG cells can be selectively abolished by GBP without interrupting spike propagation. The results suggest that the inhibitory effect of GBP on SMPO might be one of the membrane mechanisms of action of GBP. This may partially explain the antinociceptive action of GBP by directly suppression nociceptive afferent input to the spinal cord.

Protein kinase A modulates spontaneous activity in chronically compressed dorsal root ganglion neurons in the rat

&NA; Protein kinase A (PKA) can play a critical role in the modulation of neuronal excitability. We examined the role of PKA in the modulation of abnormal spontaneous activity (SA) originating from the chronically compressed dorsal root ganglion (CCD). The L4 and L5 dorsal root ganglia (DRGs) were compressed by inserting a stainless steel rod into each corresponding intervertebral foramen. After 1–14 postoperative days, SA in DRG neurons with myelinated axons was recorded in vitro from teased dorsal root microfilaments. Rp‐cAMPS (5–500 &mgr;M), a specific inhibitor of PKA, caused a dose‐dependent decrease in the discharge rate of SA when topically applied to the DRG. The highest dose completely blocked the SA, but not the conduction of action potentials. H89 (10 &mgr;M), another PKA inhibitor, also markedly decreased SA. Sp‐cAMPS (500 &mgr;M), a specific activator of PKA, increased the discharge rate of SA in all injured units tested, but did not trigger firing in silent neurons. Okadaic acid (0.1 &mgr;M), a protein phosphatase inhibitor, and forskolin (1 &mgr;M), an adenyl cyclase activator, each significantly increased the discharge rate of SA. These results strongly suggest that PKA modulates the SA in injured DRG neurons with myelinated axons.