Suppression of network activity in dorsal horn by gabapentin permeation of TRPV1 channels: implications for drug access to cytoplasmic targets.

The effectiveness of gabapentin (GBP) in the treatment of neuropathic pain depends on access to the α2δ-1 accessory subunit of voltage-gated Ca(2+) channels. Access may be limited by its rate of entry via the neuronal system L-neutral amino acid transporter. The open pore of capsaicin-activated TRPV1 channel admits organic molecules such as local anesthetics and we calculated that GBP entry via this route would be 500× more rapid than via the transporter. Capsaicin should therefore increase GBP effectiveness. We used a quaternary GBP derivative (Q-GBP) as sole charge carrier in whole-cell recording experiments on rat dorsal root ganglion (DRG) neurons. Under these conditions, capsaicin produced a capsazepine-sensitive inward current thereby confirming Q-GBP permeation of TRPV1 channels. We have previously established that 5-6 days exposure to 100 µM GBP decreases excitability of dorsal horn neurons whereas 10 µM is ineffective. Excitability was monitored using confocal Ca(2+) imaging of rat spinal cord slices in organotypic culture. GBP effectiveness was augmented by transient exposures of cultures to capsaicin and robust suppression of excitability was seen with 10 µM GBP. Experiments with an inhibitor of the neutral amino acid transporter, 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid (BCH 300 µM), showed the actions of GBP seen in the presence of capsaicin were independent of its entry by this route. Capsaicin potentiation of GBP depression of dorsal horn activity may therefore reflect drug permeation of TRPV1 channels. Agonist activation of TRP channels may provide a means for improving drug access to cytoplasmic targets in selective neuronal populations defined on the basis of type of TRP channel expressed.

Analysis of the long-term actions of gabapentin and pregabalin in dorsal root ganglia and substantia gelatinosa.

The α2δ-ligands pregabalin (PGB) and gabapentin (GBP) are used to treat neuropathic pain. We used whole cell recording to study their long-term effects on substantia gelatinosa and dorsal root ganglion (DRG) neurons. Spinal cord slices were prepared from embryonic day 13 rat embryos and maintained in organotypic culture for >5 wk (neuronal age equivalent to young adult rats). Exposure of similarly aged DRG neurons (dissociated and cultured from postnatal day 19 rats) to GBP or PGB for 5-6 days attenuated high-voltage-activated calcium channel currents (HVA ICa). Strong effects were seen in medium-sized and in small isolectin B4-negative (IB4-) DRG neurons, whereas large neurons and small neurons that bound isolectin B4 (IB4+) were hardly affected. GBP (100 µM) or PGB (10 µM) were less effective than 20 µM Mn(2+) in suppression of HVA ICa in small DRG neurons. By contrast, 5-6 days of exposure to these α2δ-ligands was more effective than 20 µM Mn(2+) in reducing spontaneous excitatory postsynaptic currents at synapses in substantia gelatinosa. Spinal actions of gabapentinoids cannot therefore be ascribed to decreased expression of HVA Ca(2+) channels in primary afferent nerve terminals. In substantia gelatinosa, 5-6 days of exposure to PGB was more effective in inhibiting excitatory synaptic drive to putative excitatory neurons than to putative inhibitory neurons. Although spontaneous inhibitory postsynaptic currents were also attenuated, the overall long-term effect of α2δ-ligands was to decrease network excitability as monitored by confocal Ca(2+) imaging. We suggest that selective actions of α2δ-ligands on populations of DRG neurons may predict their selective attenuation of excitatory transmission onto excitatory vs. inhibitory neurons in substantia gelatinosa.

Is BDNF sufficient for information transfer between microglia and dorsal horn neurons during the onset of central sensitization?

Peripheral nerve injury activates spinal microglia. This leads to enduring changes in the properties of dorsal horn neurons that initiate central sensitization and the onset of neuropathic pain. Although a variety of neuropeptides, cytokines, chemokines and neurotransmitters have been implicated at various points in this process, it is possible that much of the information transfer between activated microglia and neurons, at least in this context, may be explicable in terms of the actions of brain derived neurotrophic factor (BDNF). Microglial-derived BDNF mediates central sensitization in lamina I by attenuating inhibitory synaptic transmission. This involves an alteration in the chloride equilibrium potential as a result of down regulation of the potassium-chloride exporter, KCC2. In lamina II, BDNF duplicates many aspects of the effects of chronic constriction injury (CCI) of the sciatic nerve on excitatory transmission. It mediates an increase in synaptic drive to putative excitatory neurons whilst reducing that to inhibitory neurons. CCI produces a specific pattern of changes in excitatory synaptic transmission to tonic, delay, phasic, transient and irregular neurons. A very similar 'injury footprint' is seen following long-term exposure to BDNF. This review presents new information on the action of BDNF and CCI on lamina II neurons, including the similarity of their actions on the kinetics and distributions of subpopulations of miniature excitatory postsynaptic currents (mEPSC). These findings raise the possibility that BDNF functions as a final common path for a convergence of perturbations that culminate in the generation of neuropathic pain.

Brain-derived neurotrophic factor drives the changes in excitatory synaptic transmission in the rat superficial dorsal horn that follow sciatic nerve injury.

Peripheral nerve injury can promote neuropathic pain. The basis of the 'central sensitization' that underlies this often intractable condition was investigated using 14-20-day chronic constriction injury (CCI) of the sciatic nerve of 20-day-old rats followed by electrophysiological analysis of acutely isolated spinal cord slices. In addition, defined-medium organotypic spinal cord slice cultures were exposed for 5-6 days to brain-derived neurotrophic factor (BDNF, 200 ng ml(-1)) or to medium conditioned with activated microglia (aMCM). Since microglial activation is an early consequence of CCI, the latter manipulation allowed us to model the effect of peripheral nerve injury on the dorsal horn in vitro. Using whole-cell recording from superficial dorsal horn neurons, we found that both BDNF and CCI increased excitatory synaptic drive to putative excitatory 'radial delay' neurons and decreased synaptic excitation of inhibitory 'tonic islet/central' neurons. BDNF also attenuated synaptic excitation of putative GABAergic neurons identified by glutamic acid decarboxylase (GAD) immunoreactivity. Intrinsic neuronal properties (rheobase, input resistance and action potential discharge rates) were unaffected. Exposure of organotypic cultures to either BDNF or aMCM increased overall excitability of the dorsal horn, as seen by increased cytoplasmic Ca(2+) responses to 35 mm K(+) as monitored by confocal Fluo-4AM imaging. The effect of aMCM was attenuated by the recombinant BDNF binding protein TrkBd5 and the effect of BDNF persisted when GABAergic inhibition was blocked with SR95531. These findings suggest that CCI enhances excitatory synaptic drive to excitatory neurons but decreases that to inhibitory neurons. Both effects are mediated by nerve injury-induced release of BDNF from microglia.

P2X(3) expression is not altered by lingual nerve injury.

We have investigated a possible role for the ATP receptor subunit P2X(3), in the development of neuropathic pain following injury to a peripheral branch of the trigeminal nerve. In nine anaesthetised adult ferrets the left lingual nerve was sectioned and recovery permitted for 3 days, 3 weeks or 3 months (3 ferrets per group). A retrograde tracer, fluorogold, was applied to the nerve to allow identification of cell bodies in the trigeminal ganglion with axons in the injured nerve. Indirect immunofluorescence for P2X(3) and image analysis was used to quantify the percentage area of staining at the site of injury. Additionally, the proportion of fluorogold-positive cells that expressed P2X(3) was determined and compared with expression in non-fluorogold containing cells in another part of the ganglion. Comparisons were made with results from control animals that only received the tracer injection. After lingual nerve injury there was no significant change in P2X(3) expression at the site of nerve injury or within cell bodies linked to either injured (lingual) or uninjured (ophthalmic) axons, at any of the time periods investigated. Overall, this study suggests that P2X(3) expression at these sites is not involved in the development of neuropathic pain following lingual nerve injury.

Effect of SB-750364, a specific TRPV1 receptor antagonist, on injury-induced ectopic discharge in the lingual nerve.

Abnormal neural activity generated at a site of nerve injury is thought to contribute to the development of dysaesthesia. Vanilloid receptor 1 (TRPV1), a transducer of noxious stimuli, may be involved in the initiation of this abnormal activity and could provide a useful therapeutic target. We investigated the effect of a specific TRPV1 antagonist (SB-750364) on injury-induced discharge in the lingual nerve. In 12 anaesthetised adult ferrets the left lingual nerve was sectioned and animals were allowed to recover for 3-7 days. In terminal experiments under general anaesthesia, the nerve was re-exposed and electrophysiological recordings made from spontaneously active axons in fine filaments dissected from the nerve central to both the injury site and the junction with the chorda tympani. SB-750364 was infused via the cephalic vein in order to achieve three increasing but stable systemic blood levels of the compound (0.3, 1.0 and 3.0 microM). Twenty-eight spontaneously active units were studied, with discharge frequencies ranging from 0.02 to 4.9 Hz. There was a significant reduction in spontaneous activity in 17 units (61%) at 1.0 microM or less of SB-750364 (p<0.01; Friedman test with Dunn's multiple comparisons). A further 4 units (14%) showed a significant reduction in activity at 3.0 microM (p<0.01). In the remaining 7 units (25%) the discharge was unaffected (p>0.05). These data show that the TRPV1 antagonist SB-750364 can reduce the level of spontaneous activity initiated in some axons following lingual nerve injury.

Changes in vanilloid receptor 1 (TRPV1) expression following lingual nerve injury.

We have investigated a possible role for vanilloid receptor 1 (TRPV1), a transducer of noxious stimuli, in the development of neuropathic pain following injury to a peripheral branch of the trigeminal nerve. In nine adult ferrets the left lingual nerve was sectioned and recovery permitted for 3 days, 3 weeks or 3 months (3 ferrets per group). A retrograde tracer, fluorogold, was injected into the damaged nerve to identify associated cell bodies in the trigeminal ganglion. Three further ferrets, receiving only tracer injection, served as uninjured controls. Indirect immunofluorescence for TRPV1 and image analysis was used to quantify the percentage area of staining (PAS) of TRPV1 in the left and right lingual nerves. Additionally, the proportion of fluorogold positive and fluorogold negative cells expressing TRPV1 in the ganglion was determined. TRPV1 expression increased significantly at the injury site of damaged nerves 3 days after injury and this was matched by a reduction in the proportion of fluorogold positive cells expressing TRPV1 in the ganglion. At 3 weeks TRPV1 expression at the injury site was still high, while in the ganglion was significantly greater than in the controls. In the 3-month recovery group TRPV1 expression in both nerve fibres and ganglion cells, was not significantly different from controls and there were no changes in expression in the fluorogold negative cells in the ganglion at any time point studied. These data suggest that after injury there is an increase in the axonal transport of TRPV1 from the cell bodies to the damaged axons and this is followed by an increase in synthesis in the ganglion. These changes in expression may be involved in development of sensory disturbances or dysaesthesia after injury.

Vanilloid receptor 1 (TRPV1) expression in lingual nerve neuromas from patients with or without symptoms of burning pain.

The lingual nerve, a peripheral branch of the trigeminal nerve, can be damaged during the surgical removal of lower third molar teeth. This damage can lead to the development of dysaesthesia, with some patients complaining of burning pain. We investigated the hypothesis that vanilloid receptor 1 (TRPV1), a transducer of noxious heat stimuli, was involved in the development of this burning pain. Neuroma specimens were obtained from patients undergoing microsurgical repair of a damaged lingual nerve. Repair was undertaken where there was little evidence of spontaneous recovery, 7-41 months after the initial injury. Preoperatively the incidence of dysaesthesia was determined by reported symptoms and using visual analogue scales (VAS) for pain, tingling and discomfort. Nine neuromas were studied from patients with burning dysaesthesia and six from patients with a sensory deficit but no dysaesthesia. Indirect immunofluorescence for protein gene product (PGP) 9.5 and TRPV1 was used to quantify the percentage area of PGP 9.5 positive neuronal tissue that also expressed TRPV1. The results showed no significant difference between the mean percentage area of TRPV1 expression in neuromas from patients with or without burning dysaesthesia. Furthermore, there was no correlation between TRPV1 expression and the VAS scores for pain, tingling or discomfort. However, if data from all patients was pooled, there was a negative correlation between the level of TRPV1 expression and the time after initial injury. These data do not rule out involvement of TRPV1 in the aetiology of burning dysaesthesia following lingual nerve injury but suggest that TRPV1 at the injury site does not play a primary role.