Activation characteristics of transient receptor potential ankyrin 1 and its role in nociception.

Transient receptor potential (TRP) ankyrin 1 (TRPA1) is a Ca(2+)-permeant, nonselective cationic channel. It is predominantly expressed in the C afferent sensory nerve fibers of trigeminal and dorsal root ganglion neurons and is highly coexpressed with the nociceptive ion channel transient receptor potential vanilloid 1 (TRPV1). Several physical and chemical stimuli have been shown to activate the channel. In this study, we have used electrophysiological techniques and behavioral models to characterize the properties of TRPA1. Whole cell TRPA1 currents induced by brief application of lower concentrations of N-methyl maleimide (NMM) or allyl isothiocyanate (AITC) can be reversed readily by washout, whereas continuous application of higher concentrations of NMM or AITC completely desensitized the currents. The deactivation and desensitization kinetics differed between NMM and AITC. TRPA1 current amplitude increased with repeated application of lower concentrations of AITC, whereas saturating concentrations of AITC induced tachyphylaxis, which was more pronounced in the presence of extracellular Ca(2+). The outward rectification exhibited by native TRPA1-mediated whole cell and single-channel currents was minimal as compared with other TRP channels. TRPA1 currents were negatively modulated by protons and polyamines, both of which activate the heat-sensitive channel, TRPV1. Interestingly, neither protein kinase C nor protein kinase A activation sensitized AITC-induced currents, but each profoundly sensitized capsaicin-induced currents. Current-clamp experiments revealed that AITC produced a slow and sustained depolarization as compared with capsaicin. TRPA1 is also expressed at the central terminals of nociceptors at the caudal spinal trigeminal nucleus. Activation of TRPA1 in this area increases the frequency and amplitude of miniature excitatory or inhibitory postsynaptic currents. In behavioral studies, intraplantar and intrathecal administration of AITC induced more pronounced and prolonged changes in nociceptive behavior than those induced by capsaicin. In conclusion, the characteristics of TRPA1 we have delineated suggest that it might play a unique role in nociception.

Latency to First Event is Shorter in Psychogenic Non-epileptic Seizures than in Epileptic Seizures in an Epilepsy Monitoring Unit.

Objective: The objective was to study latency to first event among patients with psychogenic nonepileptic seizures compared (PNES) to epileptic seizures (ES) in an epilepsy monitoring unit (EMU). Introduction: PNES are common imitators of ES. This study investigates latency to first event in patients with PNES compared to patients with ES. Methods: We performed a retrospective chart review of patients admitted to our EMU from March 2016 to October 2017. We identified patients with PNES and ES. Patients with other nonepileptic events and mixed PNES (epilepsy plus PNES) were excluded. Patient demographics, baseline seizure frequency, length of EMU stay and time from admission to first event were recorded. Results: In total, 111 patients with PNES and 121 patients with ES were included. The mean age (in years) was 42 and 38, respectively. The average baseline seizure frequency was four times higher in the PNES group than the ES group. Greater than half (52%) of the patients with PNES and about one third (38%) of the patients with ES had an event within the first 24 hours. The average time to first event was 20.88 hours for the PNES group and 30.99 hours for the ES group (p<0.01). The median latency to first event was 14 hours for the PNES group and 23 hours for the ES group. The average length of EMU stay was significantly longer in the ES group (70.82 hours) than the PNES group (53.95 hours). Conclusion: The average time to first event is shorter for PNES than in ES. In patients with high pre-EMU clinical suspicion for PNES, relatively shorter EMU monitoring (24 to 48 hours) can confirm diagnosis. This phenomenon might improve cost-effectiveness of EMU monitoring in patients with PNES.

A reexamination of poneratoxin from the venom of the bullet ant Paraponera clavata.

In 1991, Piek et al. [45] described a voltage-gated sodium channel (VGSC) modifier from "bullet ant" (Paraponera clavata) venom they called poneratoxin (PoTx). Using UV chromatography and Edman degradation they showed two "identical peptides" of 25 residues. We reinvestigated PoTx using ultra performance liquid chromatography-tandem mass spectrometry (UPLC-TMS). De novo sequencing showed the two peptides were actually structurally different peptides: the originally described PoTx and a glycyl pro-peptide (glycyl-PoTx) that lacks C-terminus amidation. We examined P. clavata venom from different geographical locations and discovered two additional PoTx analogs: an A23E substitution analog and a D22N; A23V substitutions analog. We tested PoTx and these three natural analogs on the mammalian sensory voltage-gated sodium channel, Nav1.7, using whole cell voltage-clamp. PoTx and each analog induced slowly activating currents in response to small depolarizing steps and sustained currents due to blockade of channel inactivation, similar to that described previously in skeletal muscle [19]. Glycyl-PoTx had the same potency and efficacy as PoTx. A23E PoTx, with a decrease in both C-terminal net positive charge and hydrophobicity, had an eight-fold reduction in potency compared to PoTx. In contrast, the D22N; A23V PoTx, with an increase in both C-terminal net positive charge and hydrophobicity, had a nearly five-fold increase in potency compared to PoTx. We found that changes in PoTx C-terminus caused a significant change in PoTx potency.

Relationship between high-frequency oscillations and spikes in a case of temporal lobe epilepsy.

OBJECTIVE: The aim of this case report was to study the relationship between high-frequency oscillations (HFOs), spikes, and seizures in a patient with temporal lobe epilepsy. INTRODUCTION: During intracranial electroencephalography (EEG), HFOs are thought to be a marker for the seizure onset zone (SOZ). High-frequency oscillations are classified into ripples with frequencies of 70-200 Hz and fast ripples with frequencies of 200-500 Hz. Although HFOs are thought to be a marker for the SOZ, their relationship to spikes has not been studied in detail, especially within the SOZ. METHODS: We studied the time course of ripples and spikes in a patient undergoing intracranial EEG. Medications were discontinued on day one. She suffered three seizures on day three. Her SOZ was in the left hippocampus, which displayed abundant ripples and spikes. Ripples, spikes with simultaneous ripples, and spikes without ripples were counted for this study. RESULTS: We found that ripples and spikes in the SOZ had a marked diurnal variation. Ripples, spikes with ripples, and spikes without ripples increased and decreased in concert until just before seizure onset, when ripples and spikes with ripples increased markedly. Spikes without ripples did not increase. CONCLUSIONS: These results support ripples as a marker for SOZ and show that they co-occur with spikes. Seizure onset was heralded by an increase in ripples and spikes with ripples, without an increase in spikes without ripples. We hypothesize that spikes associated with ripples may have a somewhat different pathophysiological mechanism than spikes not associated with ripples, differences that may be relevant for the timing of seizure onset.

Benzonatate inhibition of voltage-gated sodium currents.

Benzonatate was FDA-approved in 1958 as an antitussive. Its mechanism of action is thought to be anesthesia of vagal sensory nerve fibers that mediate cough. Vagal sensory neurons highly express the Nav1.7 subtype of voltage-gated sodium channels, and inhibition of this channel inhibits the cough reflex. Local anesthetics inhibit voltage-gated sodium channels, but there are no reports of whether benzonatate affects these channels. Our hypothesis is that benzonatate inhibits Nav1.7 voltage-gated sodium channels. We used whole cell voltage clamp recording to test the effects of benzonatate on voltage-gated sodium (Na(+)) currents in two murine cell lines, catecholamine A differentiated (CAD) cells, which express primarily Nav1.7, and N1E-115, which express primarily Nav1.3. We found that, like local anesthetics, benzonatate strongly and reversibly inhibits voltage-gated Na(+) channels. Benzonatate causes both tonic and phasic inhibition. It has greater effects on channel inactivation than on activation, and its potency is much greater at depolarized potentials, indicating inactivated-state-specific effects. Na(+) currents in CAD cells and N1E-115 cells are similarly affected, indicating that benzonatate is not Na(+) channel subtype-specific. Benzonatate is a mixture of polyethoxy esters of 4-(butylamino) benzoic acid having varying degrees of hydrophobicity. We found that Na(+) currents are inhibited most potently by a benzonatate fraction containing the 9-ethoxy component. Detectable effects of benzonatate occur at concentrations as low as 0.3 µM, which has been reported in humans. We conclude that benzonatate has local anesthetic-like effects on voltage-gated sodium channels, including Nav1.7, which is a possible mechanism for cough suppression by the drug.

Increased neurogenesis in dentate gyrus of long-lived Ames dwarf mice.

Neurogenesis occurs throughout adult life in the dentate gyrus of mammalian hippocampus and has been suggested to play an important role in cognitive function. Multiple trophic factors including IGF-I have been demonstrated to regulate hippocampal neurogenesis. Ames dwarf mice live considerably longer than normal animals and maintain physiological function at youthful levels, including cognitive function, despite a deficiency of circulating GH and IGF-I. Here we show an increase in numbers of newly generated cells [bromodeoxyuridine (BrdU) positive] and newborn neurons (neuronal nuclear antigen and BrdU positive) in the dentate gyrus of adult dwarf mice compared with normal mice using BrdU labeling. Despite the profound suppression of hippocampal GH expression, hippocampal IGF-I protein levels are up-regulated and the corresponding mRNAs are as high in Ames dwarf as in normal mice. Our results suggest that local/hippocampal IGF-I expression may have induced the increase in hippocampal neurogenesis, and increased neurogenesis might contribute to the maintenance of youthful levels of cognitive function during aging in these long-lived animals.

The effect of vagus nerve stimulation on migraines.

Vagus nerve stimulation (VNS) inhibits nociceptive behavior in animals. VNS might reduce pain in patients with VNS device implanted for intractable seizures. One case report described possible benefits on migraines. We contacted all patients who received VNS therapy for intractable epilepsy between 1993 and 1999 at Southern Illinois University, Springfield, Illinois. Patients who had concomitant chronic pain were subsequently interviewed. Pain intensity before and after VNS implantation was rated by the patient as average, worst, and least and on numeric rating scale from 1 to 10. Current pain measurements were compared to preimplantation by using Global Pain Relief Rating Scale. Of 62 patients who received VNS, 27 patients were interviewed; 4 patients had common migraine, and no other chronic pain syndromes were identified. All patients with migraine reported reductions in headache frequency and numeric rating scale score for average and least headache intensity. One patient reported complete relief of headaches. Improvement was reported to start 1 to 3 months after initiation of therapy. On Global Pain Relief Rating Scale, 1 patient reported complete pain relief, 2 reported a lot of pain relief, and 1 reported slight pain relief. Concomitant antiepileptic drugs were decreased in 3 patients and slightly increased in 1. VNS might be beneficial for prophylactic therapy of migraine.

A biochemical characterization of the major peptides from the Venom of the giant Neotropical hunting ant Dinoponera australis.

Venom from the "false tocandira"Dinoponera australis, a giant Neotropical hunting ant, paralyzes small invertebrate prey and induces a myriad of systemic effects in large vertebrates. HPLC/DAD/MS analyses revealed that the venom has over 75 unique proteinaceous components with a large diversity of properties ranging in size, hydrophobicity, and overall abundance. The six most abundant peptides, demonstrative of this diversity and hereafter referred to as Dinoponeratoxins, were de novo sequenced by exact mass precursor ion selection and Edman degradation. The smallest peptide characterized, Da-1039, is hydrophilic and has similarities to vasoactive peptides like kinin and bombesin. The two largest and most abundant peptides, Da-3105 and Da-3177, have a 92.9% identity in a 28 residue overlap and share approximately 50 of their sequence with ponericin G2 (an antimicrobial from another ponerine ant Pachycondyla goeldii). One peptide, Da-1585, is a hydrophilic cleavage product of an amphipathic peptide, Da-2501. The most hydrophobic peptide, Da-1837, is amidated (a PTM observed in one half of the major peptides) and shares homology with poneratoxin, a sodium channel modifier found in the bullet ant Paraponera clavata. This study is the first examination of potential pharmacophores from venom of the genus Dinoponera (Order: Hymenoptera).

Selective targeting of TRPV1 expressing sensory nerve terminals in the spinal cord for long lasting analgesia.

Chronic pain is a major clinical problem and opiates are often the only treatment, but they cause significant problems ranging from sedation to deadly respiratory depression. Resiniferatoxin (RTX), a potent agonist of Transient Receptor Potential Vanilloid 1 (TRPV1), causes a slow, sustained and irreversible activation of TRPV1 and increases the frequency of spontaneous excitatory postsynaptic currents, but causes significant depression of evoked EPSCs due to nerve terminal depolarization block. Intrathecal administration of RTX to rats in the short-term inhibits nociceptive synaptic transmission, and in the long-term causes a localized, selective ablation of TRPV1-expressing central sensory nerve terminals leading to long lasting analgesia in behavioral models. Since RTX actions are selective for central sensory nerve terminals, other efferent functions of dorsal root ganglion neurons can be preserved. Preventing nociceptive transmission at the level of the spinal cord can be a useful strategy to treat chronic, debilitating and intractable pain.

Three brief epileptic seizures reduce inhibitory synaptic currents, GABA(A) currents, and GABA(A)-receptor subunits.

PURPOSE: Cellular mechanisms activated during seizures may exacerbate epilepsy. gamma-Aminobutyric acid (GABA) is the major inhibitory neurotransmitter in brain, and we hypothesized that brief epileptic seizures may reduce GABA function. METHODS: We used audiogenic seizures (AGSs) in genetically epilepsy-prone rats (GEPRs) to investigate effects of seizures on GABA-mediated inhibition in the presence of epilepsy. GEPRs are uniformly susceptible to AGSs beginning at 21 postnatal days. AGSs are brief convulsions lasting approximately 20 s, and they begin in inferior colliculus (IC). We evoked three seizures in GEPRs and compared the results with those in seizure-naive GEPRs and nonepileptic Sprague-Dawley (SD) rats, the GEPR parent strain. RESULTS: Whole-cell recording in IC slices showed that GABA-mediated monosynaptic inhibitory postsynaptic currents (IPSCs) were reduced 55% by three brief epileptic seizures. Whole-cell recording in IC neuronal cultures showed that currents elicited by GABA were reduced 67% by three seizures. Western blotting for the alpha1 and alpha4 subunits of the GABA(A) receptor showed no statistically significant effects. In contrast, three brief epileptic seizures reduced gamma2 subunit levels by 80%. CONCLUSIONS: The effects of the very first seizures, in animals known to be epileptic, in an area of brain known to be critical to the seizure network, were studied. The results indicate that even brief epileptic seizures can markedly reduce IPSCs and GABA currents and alter GABA(A)-receptor subunit protein levels. The cause of the reductions in IPSCs and GABA currents is likely to be altered receptor subunit composition, with reduced gamma2 levels causing reduced GABA(A)-receptor sensitivity to GABA. Seizure-induced reductions in GABA-mediated inhibition could exacerbate epilepsy.

Monoamine uptake inhibitors block alpha7-nAChR-mediated cerebral nitrergic neurogenic vasodilation.

We have proposed that activation of cerebral perivascular sympathetic alpha7-nicotinic acetylcholine receptors (alpha7-nAChRs) by nicotinic agonists releases norepinephrine, which then acts on parasympathetic nitrergic nerves, resulting in release of nitric oxide and vasodilation. Using patch-clamp electrophysiology, immunohistochemistry, and in vitro tissue bath myography, we tested this axo-axonal interaction hypothesis further by examining whether blocking norepinephrine reuptake enhanced alpha7-nAChR-mediated cerebral nitrergic neurogenic vasodilation. The results indicated that choline- and nicotine-induced alpha7-nAChR-mediated nitrergic neurogenic relaxation in endothelium-denuded isolated porcine basilar artery rings was enhanced by desipramine and imipramine at lower concentrations (0.03-0.1 microM) but inhibited at higher concentrations (0.3-10 microM). In cultured superior cervical ganglion (SCG) neurons of the pig and rat, choline (0.1-30 mM)-evoked inward currents were reversibly blocked by 1-30 microM mecamylamine, 1-30 microM methyllycaconitine, 10-300 nM alpha-bungarotoxin, and 0.1-10 microM desipramine and imipramine, providing electrophysiological evidence for the presence of similar functional alpha7-nAChRs in cerebral perivascular sympathetic neurons of pigs and rats. In alpha7-nAChR-expressing Xenopus oocytes, choline-elicited inward currents were attenuated by alpha-bungarotoxin, imipramine, and desipramine. These monoamine uptake inhibitors appeared to directly block the alpha7-nAChR, resulting in diminished nicotinic agonist-induced cerebral nitrergic vasodilation. The enhanced nitrergic vasodilation by lower concentrations of monoamine uptake inhibitors is likely due to a greater effect on monoamine uptake than on alpha7-nAChR blockade. These results further support the hypothesis of axo-axonal interaction in nitrergic regulation of cerebral vascular tone.