Alexander M. Binshtok

GTP cyclohydrolase and tetrahydrobiopterin regulate pain sensitivity and persistence.

We report that GTP cyclohydrolase (GCH1), the rate-limiting enzyme for tetrahydrobiopterin (BH4) synthesis, is a key modulator of peripheral neuropathic and inflammatory pain. BH4 is an essential cofactor for catecholamine, serotonin and nitric oxide production. After axonal injury, concentrations of BH4 rose in primary sensory neurons, owing to upregulation of GCH1. After peripheral inflammation, BH4 also increased in dorsal root ganglia (DRGs), owing to enhanced GCH1 enzyme activity. Inhibiting this de novo BH4 synthesis in rats attenuated neuropathic and inflammatory pain and prevented nerve injury–evoked excess nitric oxide production in the DRG, whereas administering BH4 intrathecally exacerbated pain. In humans, a haplotype of the GCH1 gene (population frequency 15.4%) was significantly associated with less pain following diskectomy for persistent radicular low back pain. Healthy individuals homozygous for this haplotype exhibited reduced experimental pain sensitivity, and forskolin-stimulated immortalized leukocytes from haplotype carriers upregulated GCH1 less than did controls. BH4 is therefore an intrinsic regulator of pain sensitivity and chronicity, and the GTP cyclohydrolase haplotype is a marker for these traits.

Nociceptors are interleukin-1beta sensors.

A cardinal feature of inflammation is heightened pain sensitivity at the site of the inflamed tissue. This results from the local release by immune and injured cells of nociceptor sensitizers, including prostaglandin E2, bradykinin, and nerve growth factor, that reduce the threshold and increase the excitability of the peripheral terminals of nociceptors so that they now respond to innocuous stimuli: the phenomenon of peripheral sensitization. We show here that the proinflammatory cytokine interleukin-1β (IL-1β), in addition to producing inflammation and inducing synthesis of several nociceptor sensitizers, also rapidly and directly activates nociceptors to generate action potentials and induce pain hypersensitivity. IL-1β acts in a p38 mitogen-activated protein kinase (p38 MAP kinase)-dependent manner, to increase the excitability of nociceptors by relieving resting slow inactivation of tetrodotoxin-resistant voltage-gated sodium channels and also enhances persistent TTX-resistant current near threshold. By acting as an IL-1β sensor, nociceptors can directly signal the presence of ongoing tissue inflammation.

Inhibition of nociceptors by TRPV1-mediated entry of impermeant sodium channel blockers.

Most local anaesthetics used clinically are relatively hydrophobic molecules that gain access to their blocking site on the sodium channel by diffusing into or through the cell membrane. These anaesthetics block sodium channels and thereby the excitability of all neurons, not just sensory neurons. We tested the possibility of selectively blocking the excitability of primary sensory nociceptor (pain-sensing) neurons by introducing the charged, membrane-impermeant lidocaine derivative QX-314 through the pore of the noxious-heat-sensitive TRPV1 channel. Here we show that charged sodium-channel blockers can be targeted into nociceptors by the application of TRPV1 agonists to produce a pain-specific local anaesthesia. QX-314 applied externally had no effect on the activity of sodium channels in small sensory neurons when applied alone, but when applied in the presence of the TRPV1 agonist capsaicin, QX-314 blocked sodium channels and inhibited excitability. Inhibition by co-applied QX-314 and capsaicin was restricted to neurons expressing TRPV1. Injection of QX-314 together with capsaicin into rat hindpaws produced a long-lasting (more than 2 h) increase in mechanical and thermal nociceptive thresholds. Long-lasting decreases in pain sensitivity were also seen with regional injection of QX-314 and capsaicin near the sciatic nerve; however, in contrast to the effect of lidocaine, the application of QX-314 and capsaicin together was not accompanied by motor or tactile deficits.

BACE1 regulates voltage-gated sodium channels and neuronal activity

BACE1 activity is significantly increased in the brains of Alzheimers disease patients, potentially contributing to neurodegeneration. The voltage-gated sodium channel (Nav1) β2-subunit (β2), a type I membrane protein that covalently binds to Nav1 α-subunits, is a substrate for BACE1 and γ-secretase. Here, we find that BACE1–γ-secretase cleavages release the intracellular domain of β2, which increases mRNA and protein levels of the pore-forming Nav1.1 α-subunit in neuroblastoma cells. Similarly, endogenous β2 processing and Nav1.1 protein levels are elevated in brains of BACE1-transgenic mice and Alzheimers disease patients with high BACE1 levels. However, Nav1.1 is retained inside the cells and cell surface expression of the Nav1 α-subunits and sodium current densities are markedly reduced in both neuroblastoma cells and adult hippocampal neurons from BACE1-transgenic mice. BACE1, by cleaving β2, thus regulates Nav1 α-subunit levels and controls cell-surface sodium current densities. BACE1 inhibitors may normalize membrane excitability in Alzheimers disease patients with elevated BACE1 activity.

Voltage-gated sodium channels in pain states: Role in pathophysiology and targets for treatment

Pain is a major unmet medical need which has been causally linked to changes in sodium channel expression, modulation, or mutations that alter channel gating properties or current density in nociceptor neurons. Voltage-gated sodium channels activate (open) then rapidly inactivate in response to a depolarization of the plasma membrane of excitable cells allowing the transient flow of sodium ions thus generating an inward current which underlies the generation and conduction of action potentials (AP) in these cells. Activation and inactivation, as well as other gating properties, of sodium channel isoforms have different kinetics and voltage-dependent properties, so that the ensemble of channels that are present determine the electrogenic properties of specific neurons. Biophysical and pharmacological studies have identified the peripheral-specific sodium channels Na(v)1.7, Na(v)1.8 and Na(v)1.9 as particularly important in the pathophysiology of different pain syndromes, and isoform-specific blockers of these channels or targeting their modulators hold the promise of a future effective therapy for treatment of pain.

Functionally Distinct NMDA Receptors Mediate Horizontal Connectivity within Layer 4 of Mouse Barrel Cortex

In sensory areas of neocortex, thalamocortical afferents project primarily onto the spiny stellate neurons of Layer 4. Anatomical evidence indicates that these cells receive most of their excitatory input from other cortical neurons, including other spiny stellate cells. Although this local network must play an important role in sensory processing, little is known about the properties of the neurons and synapses involved. We have produced a slice preparation of mouse barrel cortex that isolates Layer 4. We report that excitatory interaction between spiny stellate neurons is largely via N-methyl-D-aspartate receptors (NMDARs) and that a given neuron contains more than one type of NMDAR, as distinguished by voltage dependence. Thus, spiny stellate cells act as effective integrators of powerful and persistent NMDAR-mediated recurrent excitation.

Activity-dependent silencing reveals functionally distinct itch-generating sensory neurons

The peripheral terminals of primary sensory neurons detect histamine and non-histamine itch-provoking ligands through molecularly distinct transduction mechanisms. It remains unclear, however, whether these distinct pruritogens activate the same or different afferent fibers. Using a strategy of reversibly silencing specific subsets of murine pruritogen-sensitive sensory axons by targeted delivery of a charged sodium-channel blocker, we found that functional blockade of histamine itch did not affect the itch evoked by chloroquine or SLIGRL-NH2, and vice versa. Notably, blocking itch-generating fibers did not reduce pain-associated behavior. However, silencing TRPV1+ or TRPA1+ neurons allowed allyl isothiocyanate or capsaicin, respectively, to evoke itch, implying that certain peripheral afferents may normally indirectly inhibit algogens from eliciting itch. These findings support the presence of functionally distinct sets of itch-generating neurons and suggest that targeted silencing of activated sensory fibers may represent a clinically useful anti-pruritic therapeutic approach for histaminergic and non-histaminergic pruritus.

Coapplication of Lidocaine and the Permanently Charged Sodium Channel Blocker QX-314 Produces a Long-lasting Nociceptive Blockade in Rodents

Background:Nociceptive-selective local anesthesia is produced by entry of the permanently charged lidocaine-derivative QX-314 into nociceptors when coadministered with capsaicin, a transient receptor potential vanilloid 1 (TRPV1) channel agonist. However, the pain evoked by capsaicin before establishment of the QX-314–mediated block would limit clinical utility. Because TRPV1 channels are also activated by lidocaine, the authors tested whether lidocaine can substitute for capsaicin to introduce QX-314 into nociceptors through TRPV1 channels and produce selective analgesia. Methods:Lidocaine (0.5% [17.5 mm], 1% [35 mm], and 2% [70 mm]) alone, QX-314 (0.2% [5.8 mm]) alone, and a combination of the two were injected subcutaneously and adjacent to the sciatic nerve in rats and mice. Mechanical and thermal responsiveness were measured, as was motor block. Results:Coapplication of 0.2% QX-314 with lidocaine prolonged the nociceptive block relative to lidocaine alone, an effect attenuated in TRPV1 knockout mice. The 0.2% QX-314 alone had no effect when injected intraplantary or perineurally, and it produced only weak short-lasting inhibition of the cutaneous trunci muscle reflex. Perisciatic nerve injection of lidocaine with QX-314 produced a differential nociceptive block much longer than the transient motor block, lasting 2 h (for 1% lidocaine) to 9 h (2% lidocaine). Triple application of lidocaine, QX-314, and capsaicin further increased the duration of the differential block. Conclusions:Coapplication of lidocaine and its quaternary derivative QX-314 produces a long-lasting, predominantly nociceptor-selective block, likely by facilitating QX-314 entry through TRPV1 channels. Delivery of QX-314 into nociceptors by using lidocaine instead of capsaicin produces sustained regional analgesia without nocifensive behavior.

NMDA Receptors in Layer 4 Spiny Stellate Cells of the Mouse Barrel Cortex Contain the NR2C Subunit

In layer 4 of the somatosensory cortex, the glutamatergic synapses that interconnect spiny stellate (SpS) neurons, which are the major targets of thalamocortical input, differ from most other neocortical excitatory synapses in that they have an extremely large NMDA receptor (NMDAR)-mediated component that is relatively insensitive to voltage-dependent Mg2+ blockade. We now report that this unique feature of the NMDA response reflects the distinctive subunit composition of the underlying receptors. We studied NMDAR-mediated miniature EPSCs (mEPSCs) and NMDA channel currents in tangential brain slices of mouse barrel cortex, which exclusively contain layer 4. NMDAR-mediated mEPSCs in SpS neurons were prominent at negative membrane potentials, and NMDA channels in outside-out patches excised from the somata of the same neurons had relatively low conductance and reduced susceptibility to Mg2+ block. These are characteristic features of heteromeric NMDAR assemblies that contain the NR2C subunit. Some patches also contained NMDA channels with higher conductance and a greater sensitivity to Mg2+. In the neocortex of transgenic mice in which a β-galactosidase (lacZ) indicator gene was controlled by the NR2C promoter, the lacZ indicator was densely expressed in layer 4. In current-clamp recordings, blockade of NMDARs caused hyperpolarization and an increase in apparent input resistance. Our data demonstrate that the SpS neurons of layer 4 functionally express NR2C subunits; this is the likely explanation for their ability to generate large NMDAR-mediated EPSPs that are effective at resting potential, without previous depolarization.

Targeting of sodium channel blockers into nociceptors to produce long‐duration analgesia: a systematic study and review

BACKGROUND AND PURPOSE We have developed a strategy to target the permanently charged lidocaine derivative lidocaine N‐ethyl bromide (QX‐314) selectively into nociceptive sensory neurons through the large‐pore transient receptor potential cation channel subfamily V (TRPV1) noxious heat detector channel. This involves co‐administration of QX‐314 and a TRPV1 agonist to produce a long‐lasting local analgesia. For potential clinical use we propose using lidocaine as the TRPV1 agonist, because it activates TRPV1 at clinical doses.