Xiu-Lin Zhang

Calcium Channel α2δ1 Subunit Mediates Spinal Hyperexcitability in Pain Modulation

&NA; Mechanisms of chronic pain, including neuropathic pain, are poorly understood. Upregulation of voltage‐gated calcium channel (VGCC) &agr;2&dgr;1 subunit (Cav&agr;2&dgr;1) in sensory neurons and dorsal spinal cord by peripheral nerve injury has been suggested to contribute to neuropathic pain. To investigate the mechanisms without the influence of other injury factors, we have created transgenic mice that constitutively overexpress Cav&agr;2&dgr;1 in neuronal tissues. Cav&agr;2&dgr;1 overexpression resulted in enhanced currents, altered kinetics and voltage‐dependence of VGCC activation in sensory neurons; exaggerated and prolonged dorsal horn neuronal responses to mechanical and thermal stimulations at the periphery; and pain behaviors. However, the transgenic mice showed normal dorsal horn neuronal responses to windup stimulation, and behavioral responses to tissue‐injury/inflammatory stimuli. The pain behaviors in the transgenic mice had a pharmacological profile suggesting a selective contribution of elevated Cav&agr;2&dgr;1 to the abnormal sensations, at least at the spinal cord level. In addition, gabapentin blocked VGCC currents concentration‐dependently in transgenic, but not wild‐type, sensory neurons. Thus, elevated neuronal Cav&agr;2&dgr;1 contributes to specific pain states through a mechanism mediated at least partially by enhanced VGCC activity in sensory neurons and hyperexcitability in dorsal horn neurons in response to peripheral stimulation. Modulation of enhanced VGCC activity by gabapentin may underlie at least partially its antihyperalgesic actions.

Intracellular calcium regulation among subpopulations of rat dorsal root ganglion neurons

Primary afferent neurons are functionally heterogeneous. To determine whether this functional heterogeneity reflects, in part, heterogeneity in the regulation of the concentration of intracellular Ca2+ ([Ca2+]i), the magnitude and decay of evoked Ca2+ transients were assessed in subpopulations of dorsal root ganglion (DRG) neurons with voltage clamp and fura‐2 ratiometric imaging. To determine whether differences in evoked Ca2+ transients among subpopulations of DRG neurons reflected differences in the contribution of Ca2+ regulatory mechanisms, pharmacological techniques were employed to assess the contribution of influx, efflux, release and uptake pathways. Subpopulations of DRG neurons were defined by cell body size, binding of the plant lectin IB4 and responsiveness to the algogenic compound capsaicin (CAP). Ca2+ transients were evoked with 30 mm K+ or voltage steps to 0 mV. There were marked differences between subpopulations of neurons with respect to both the magnitude and decay of the Ca2+ transient, with the largest and most slowly decaying Ca2+ transients in small‐diameter, IB4‐positive, CAP‐responsive neurons. The smallest and most rapidly decaying transients were in large‐diameter, IB4‐negative and CAP‐unresponsive DRG neurons. These differences were not due to a differential distribution of voltage‐gated Ca2+ currents. However, these differences did appear to reflect a differential contribution of other influx, efflux, release and uptake mechanisms between subpopulations of neurons. These results suggest that electrical activity in subpopulations of DRG neurons will have a differential influence on Ca2+‐regulated phenomena such as spike adaptation, transmitter release and gene transcription. Significantly more activity should be required in large‐diameter non‐nociceptive afferents than in small‐diameter nociceptive afferents to have a comparable influence on these processes.

High glucose-induced expression of inflammatory cytokines and reactive oxygen species in cultured astrocytes.

Astrocyte activation plays important roles both in physiological and pathological process in the CNS. In the latter, the process is further aggravated by hyperglycemia, leading to diabetes complications of CNS. We report here that high glucose (HG) treatment stimulated astrocytic morphological alteration coupled with changes in glial fibrillary acidic protein (GFAP) and vimentin expression. Additionally, HG upregulated the expression of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), interleukin-1β (IL-1β), interleukin-4 (IL-4), and vascular endothelial growth factor (VEGF); however, its effects on transforming growth factor-β (TGF-β) expression were not evident. HG treatment induced increased production of reactive oxygen species (ROS) as well as activation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and signal transducer and activator transcription 3 (STAT 3). HG-induced expression of TNF-α, IL-6, IL-1β, IL-4, and VEGF was blocked by ROS scavenger and inhibitors specific for NF-κB and STAT 3, respectively. The results suggest that the aforementioned multiple inflammatory cytokines and mediators that may be linked to the pathogenesis of the diabetes complications of CNS are induced by HG via the key signaling pathways.

Persistent inflammation alters the density and distribution of voltage-activated calcium channels in subpopulations of rat cutaneous DRG neurons.

&NA; The impact of persistent inflammation on voltage‐activated Ca2+ channels in cutaneous DRG neurons from adult rats was assessed with whole cell patch clamp techniques, sqRT‐PCR and Western blot analysis. Inflammation was induced with a subcutaneous injection of complete Freunds adjuvant (CFA). DiI was used to identify DRG neurons innervating the site of inflammation. Three days after CFA injection, high threshold Ca2+ current (HVA) density was significantly reduced in small and medium, but not large diameter neurons, reflecting a decrease in N‐, L‐ and P/Q‐type currents. This decrease in HVA current was associated with an increase in mRNA encoding the &agr;2&dgr;1‐subunit complex, but no detectable change in N‐type subunit (CaV2.2) mRNA. An increase in both &agr;2&dgr;1 and CaV2.2 protein was detected in the central nerves arising from L4 and L5 ganglia ipsilateral to the site of inflammation. In current clamp experiments on small and medium diameter cutaneous DRG neurons from naïve rats, blocking ˜40% of HVA current with Cd2+ (5 &mgr;M), had opposite effects on subpopulations of cutaneous DRG neurons (increasing excitability and action potential duration in some and decreasing excitability in others). The alterations in the density and distribution of voltage‐activated Ca2+ channels in subpopulations of cutaneous DRG neurons that develop following CFA injection should contribute to changes in sensory transmission observed in the presence of inflammation.

Effects of ginsenoside Rg1 or 17β-estradiol on a cognitively impaired, ovariectomized rat model of Alzheimer's disease.

Ginsenoside Rg1, which could improve spatial learning and memory, might be a useful agent for preventing and treating cognitive impairment in Alzheimers disease (AD). The present study was designed to test the neuroprotective effects of ginsenoside Rg1 on an ovariectomized (OVX) and d-galactose (d-gal)-injected rat model of AD, which is characterized with progressive learning and memory deficits, AD-related molecules alteration and differentiation/apoptosis imbalance in hippocampal neurons. OVX Wistar rats received daily injections of d-gal (100mg/kg) combined with different concentrations of ginsenoside Rg1 (5, 10, 20mg/kg) or 17-β-estradiol (E2, 100 μg/kg), or normal saline (NS, 1.0 ml/kg) for 6 weeks. Ovarian steroid deprivation plus d-gal injection led to spatial learning and memory capacity impairments, as well as increased Aβ(1-42) production. Ginsenoside Rg1 and E2-treatment significantly ameliorated these deteriorations in AD rats. Seven weeks after surgery, α-secretase a disintegrin and metallopeptidase domain 10 (ADAM 10) in hippocampus of AD rats was dramatically decreased, while β-secretase β-site APP-cleaving enzyme 1 (BACE 1) increased compared with those in sham-operated ones (P<0.05). Levels of cleaved caspase 3 were increased in the hippocampus of AD rats. Ginsenoside Rg1 and E2-treatment increased ADAM 10 level while reduced BACE 1 level and apoptosis. Moreover, moderate i.e. 10mg/kg/d and high i.e. 20mg/kg/d ginsenoside Rg1 displayed more effective function than low i.e. 5mg/kg/d ginsenoside Rg1. Our findings demonstrate the neuroprotective effects of ginsenoside Rg1 and E2 on AD rats and support the potential application of ginsenoside Rg1 in the treatment of learning and memory impairments in postmenopausal women.

BKCa currents are enriched in a subpopulation of adult rat cutaneous nociceptive dorsal root ganglion neurons.

The biophysical properties and distribution of voltage‐dependent, Ca2+ ‐modulated K+ (BKCa) currents among subpopulations of acutely dissociated DiI‐labeled cutaneous sensory neurons from the adult rat were characterized with whole‐cell patch‐clamp techniques. BKCa currents were isolated from total K+ current with iberiotoxin, charybdotoxin or paxilline. There was considerable variability in biophysical properties of BKCa currents. There was also variability in the distribution of BKCa current among subpopulations of cutaneous dorsal root ganglia (DRG) neurons. While present in each of the subpopulations defined by cell body size, IB4 binding or capsaicin sensitivity, BKCa current was present in the vast majority (> 90%) of small‐diameter IB4+ neurons, but was present in only a minority of neurons in subpopulations defined by other criteria (i.e. small‐diameter IB4−). Current‐clamp analysis indicated that in IB4+ neurons, BKCa currents contribute to the repolarization of the action potential and adaptation in response to sustained membrane depolarization, while playing little role in the determination of action potential threshold. Reverse transcriptase‐polymerase chain reaction analysis of mRNA collected from whole DRG revealed the presence of multiple splice variants of the BKCa channel α‐subunit, rslo and all four of the accessory β‐subunits, suggesting that heterogeneity in the biophysical and pharmacological properties of BKCa current in cutaneous neurons reflects, at least in part, the differential distribution of splice variants and/or β‐subunits. Because even a small decrease in BKCa current appears to have a dramatic influence on excitability, modulation of this current may contribute to sensitization of nociceptive afferents observed following tissue injury.

Dihydropyridine block of voltage-dependent K+ currents in rat dorsal root ganglion neurons

The dihydropyridines nifedipine, nimodipine and Bay K 8644 are widely used as pharmacological tools to assess the contribution of L-type voltage-gated Ca(2+) channels to a variety of neuronal processes including synaptic transmission, excitability and second messenger signaling. These compounds are still used in neuronal preparations despite evidence from cardiac tissue and heterologous expression systems that they block several voltage-dependent K(+) (Kv) channels. Both because these compounds have been used to assess the relative contribution of L-type Ca(2+) channels to several different processes in dorsal root ganglion (DRG) neurons and because a relatively wide variety of Kv channels present in other neuronal populations is present in DRG neurons, we determined the extent to which dihydropyridines block Kv currents in these neurons. Standard whole cell patch clamp techniques were used to study acutely disassociated adult rat DRG neurons. All three dihydropyridines tested blocked Kv currents in DRG neurons; IC(50) values (concentration resulting in an inhibition that is 50% of maximum) for nifedipine and nimodipine-induced block of sustained Kv currents were 14.5 and 6.6 microM, respectively. The magnitude of sustained current block was 44+/-1.6%, 60+/-2%, and 56+/-2.9% with 10 microM nifedipine, nimodipine and Bay K 8644, respectively. Current block was occluded by neither 4-aminopyridine (5 mM) nor tetraethylammonium (135 mM). Dihydropyridine-induced block of Kv currents was not associated with a shift in the voltage-dependence of current activation or inactivation, the recovery from inactivation, or voltage dependent block. However, there was a small use-dependence to the dihydropyridine-induced block. Our results suggest that several types of Kv channels in DRG neurons are blocked by mechanisms distinct from those underlying block of Kv channels in cardiac myocytes. Importantly, our results suggest that if investigators wish to explore the contribution of L-type Ca(2+) channels to neuronal function, they should consider alternative strategies for the manipulation of these channels than the use of dihydropyridines.

Voltage-gated Na+ currents in human dorsal root ganglion neurons

Available evidence indicates voltage-gated Na+ channels (VGSCs) in peripheral sensory neurons are essential for the pain and hypersensitivity associated with tissue injury. However, our understanding of the biophysical and pharmacological properties of the channels in sensory neurons is largely based on the study of heterologous systems or rodent tissue, despite evidence that both expression systems and species differences influence these properties. Therefore, we sought to determine the extent to which the biophysical and pharmacological properties of VGSCs were comparable in rat and human sensory neurons. Whole cell patch clamp techniques were used to study Na+ currents in acutely dissociated neurons from human and rat. Our results indicate that while the two major current types, generally referred to as tetrodotoxin (TTX)-sensitive and TTX-resistant were qualitatively similar in neurons from rats and humans, there were several differences that have important implications for drug development as well as our understanding of pain mechanisms. DOI: http://dx.doi.org/10.7554/eLife.23235.001

Inflammation-induced increase in nicotinic acetylcholine receptor current in cutaneous nociceptive DRG neurons from the adult rat.

The goals of the present study were to determine (1) the properties of the nicotinic acetylcholine receptor (nAChR) currents in rat cutaneous dorsal root ganglion (DRG) neurons; (2) the impact of nAChR activation on the excitability of cutaneous DRG neurons; and (3) the impact of inflammation on the density and distribution of nAChR currents among cutaneous DRG neurons. Whole-cell patch-clamp techniques were used to study retrogradely labeled DRG neurons from naïve and complete Freunds adjuvant inflamed rats. Nicotine-evoked currents were detectable in ∼70% of the cutaneous DRG neurons, where only one of two current types, fast or slow currents based on rates of activation and inactivation, was present in each neuron. The biophysical and pharmacological properties of the fast current were consistent with nAChRs containing an α7 subunit while those of the slow current were consistent with nAChRs containing α3/β4 subunits. The majority of small diameter neurons with fast current were IB4- while the majority of small diameter neurons with slow current were IB4+. Preincubation with nicotine (1 μM) produced a transient (1 min) depolarization and increase in the excitability of neurons with fast current and a decrease in the amplitude of capsaicin-evoked current in neurons with slow current. Inflammation increased the current density of both slow and fast currents in small diameter neurons and increased the percentage of neurons with the fast current. With the relatively selective distribution of nAChR currents in putative nociceptive cutaneous DRG neurons, our results suggest that the role of these receptors in inflammatory hyperalgesia is likely to be complex and dependent on the concentration and timing of acetylcholine release in the periphery.

Activity-dependent hyperpolarization of EGABA is absent in cutaneous DRG neurons from inflamed rats

A shift in GABA(A) signaling from inhibition to excitation in primary afferent neurons appears to contribute to the inflammation-induced increase in afferent input to the CNS. An activity-dependent depolarization of the GABA(A) current equilibrium potential (E(GABA)) has been described in CNS neurons which drives a shift in GABA(A) signaling from inhibition to excitation. The purpose of the present study was to determine if such an activity-dependent depolarization of E(GABA) occurs in primary afferents and whether the depolarization is amplified with persistent inflammation. Acutely dissociated retrogradely labeled cutaneous dorsal root ganglion (DRG) neurons from naïve and inflamed rats were studied with gramicidin perforated patch recording. Rather than a depolarization, 200 action potentials delivered at 2 Hz resulted in a ∼10 mV hyperpolarization of E(GABA) in cutaneous neurons from naïve rats. No such hyperpolarization was observed in neurons from inflamed rats. The shift in E(GABA) was not blocked by 10 μM bumetanide. Furthermore, because activity-dependent hyperpolarization of E(GABA) was fully manifest in the absence of HCO₃⁻ in the bath solution, this shift was not dependent on a change in HCO₃⁻-Cl⁻ exchanger activity, despite evidence of HCO₃⁻-Cl⁻ exchangers in DRG neurons that may contribute to the establishment of E(GABA) in the presence of HCO₃⁻. While the mechanism underlying the activity-dependent hyperpolarization of E(GABA) has yet to be identified, because this mechanism appears to function as a form of feedback inhibition, facilitating GABA-mediated inhibition of afferent activity, it may serve as a novel target for the treatment of inflammatory pain.