Development of KVO treatment strategies for chronic pain in a rat model of Gulf War Illness.

We examined whether combinations of Kv7 channel openers could be effective modifiers of deep tissue nociceptor activity; and whether such combinations could then be optimized for use as safe analgesics for pain-like signs that developed in a rat model of GWI (Gulf War Illness) pain. Voltage clamp experiments were performed on subclassified nociceptors isolated from rat DRG (dorsal root ganglion). A stepped voltage protocol was applied (-55 to -40 mV; Vh = -60 mV; 1500 ms) and Kv7 evoked currents were subsequently isolated by linopirdine subtraction. Directly activated and voltage activated K+ currents were characterized in the presence and absence of Retigabine (5-100 µM) and/or Diclofenac (50-140 µM). Retigabine produced substantial voltage dependent effects and a maximal sustained current of 1.14 pA/pF ± 0.15 (ED50: 62.7 ± 3.18 µM). Diclofenac produced weak voltage dependent effects but a similar maximum sustained current of 1.01 ± 0.26 pA/pF (ED50: 93.2 ± 8.99 µM). Combinations of Retigabine and Diclofenac substantially amplified resting currents but had little effect on voltage dependence. Using a cholinergic challenge test (Oxotremorine, 10 µM) associated with our GWI rat model, combinations of Retigabine (5 uM) and Diclofenac (2.5, 20 and 50 µM) substantially reduced or totally abrogated action potential discharge to the cholinergic challenge. When combinations of Retigabine and Diclofenac were used to relieve pain-signs in our rat model of GWI, only those combinations associated with serious subacute side effects could relieve pain-like behaviors.

Behavioral, cellular and molecular maladaptations covary with exposure to pyridostigmine bromide in a rat model of gulf war illness pain.

Many veterans of Operation Desert Storm (ODS) struggle with the chronic pain of Gulf War Illness (GWI). Exposure to insecticides and pyridostigmine bromide (PB) have been implicated in the etiology of this multisymptom disease. We examined the influence of 3 (DEET (N,N-diethyl-meta-toluamide), permethrin, chlorpyrifos) or 4 GW agents (DEET, permethrin, chlorpyrifos, pyridostigmine bromide (PB)) on the post-exposure ambulatory and resting behaviors of rats. In three independent studies, rats that were exposed to all 4 agents consistently developed both immediate and delayed ambulatory deficits that persisted at least 16 weeks after exposures had ceased. Rats exposed to a 3 agent protocol (PB excluded) did not develop any ambulatory deficits. Cellular and molecular studies on nociceptors harvested from 16WP (weeks post-exposure) rats indicated that vascular nociceptor Nav1.9 mediated currents were chronically potentiated following the 4 agent protocol but not following the 3 agent protocol. Muscarinic linkages to muscle nociceptor TRPA1 were also potentiated in the 4 agent but not the 3 agent, PB excluded, protocol. Although Kv7 activity changes diverged from the behavioral data, a Kv7 opener, retigabine, transiently reversed ambulation deficits. We concluded that PB played a critical role in the development of pain-like signs in a GWI rat model and that shifts in Nav1.9 and TRPA1 activity were critical to the expression of these pain behaviors.

DEET potentiates the development and persistence of anticholinesterase dependent chronic pain signs in a rat model of Gulf War Illness pain.

Exposure to DEET (N,N-diethyl-meta-toluamide) may have influenced the pattern of symptoms observed in soldiers with GWI (Gulf War Illness; Haley and Kurt, 1997). We examined how the addition of DEET (400mg/kg; 50% topical) to an exposure protocol of permethrin (2.6mg/kg; topical), chlorpyrifos (CP; 120mg/kg), and pyridostigmine bromide (PB;13mg/kg) altered the emergence and pattern of pain signs in an animal model of GWI pain (Nutter et al., 2015). Rats underwent behavioral testing before, during and after a 4week exposure: 1) hindlimb pressure withdrawal threshold; 2) ambulation (movement distance and rate); and 3) resting duration. Additional studies were conducted to assess the influence of acute DEET (10-100µM) on muscle and vascular nociceptor Kv7, KDR, Nav1.8 and Nav1.9. We report that a 50% concentration of DEET enhanced the development and persistence of pain-signs. Rats exposed to all 4 compounds exhibited ambulation deficits that appeared 5-12weeks post-exposure and persisted through weeks 21-24. Rats exposed to only three agents (CP or PB excluded), did not fully develop ambulation deficits. When PB was excluded, rats also developed rest duration pain signs, in addition to ambulation deficits. There was no evidence that physiological doses of DEET acutely modified nociceptor Kv7, KDR, Nav1.8 or Nav1.9 activities. Nevertheless, DEET augmented protocols decreased the conductance of Kv7 expressed in vascular nociceptors harvested from chronically exposed rats. We concluded that DEET enhanced the development and persistence of pain behaviors, but the anticholinesterases CP and PB played a determinant role.

Combinations of classical and non-classical voltage dependent potassium channel openers suppress nociceptor discharge and reverse chronic pain signs in a rat model of Gulf War illness.

In a companion paper we examined whether combinations of Kv7 channel openers (Retigabine and Diclofenac; RET, DIC) could be effective modifiers of deep tissue nociceptor activity; and whether such combinations could then be optimized for use as safe analgesics for pain-like signs that developed in a rat model of GWI (Gulf War Illness) pain. In the present report, we examined the combinations of Retigabine/Meclofenamate (RET/MEC) and Meclofenamate/Diclofenac (MEC/DIC). Voltage clamp experiments were performed on deep tissue nociceptors isolated from rat DRG (dorsal root ganglion). In voltage clamp studies, a stepped voltage protocol was applied (-55 to -40 mV; Vh=-60 mV; 1500 msec) and Kv7 evoked currents were subsequently isolated by Linopirdine subtraction. MEC greatly enhanced voltage dependent conductance and produced exceptional maximum sustained currents of 6.01 ± 0.26 pA/pF (EC50: 62.2 ± 8.99 µM). Combinations of RET/MEC, and MEC/DIC substantially amplified resting currents at low concentrations. MEC/DIC also greatly improved voltage dependent conductance. In current clamp experiments, a cholinergic challenge test (Oxotremorine-M, 10 µM; OXO), associated with our GWI rat model, produced powerful action potential (AP) bursts (85 APs). Optimized combinations of RET/MEC (5 and 0.5 µM) and MEC/DIC (0.5 and 2.5 µM) significantly reduced AP discharges to 3 and 7 Aps, respectively. Treatment of pain-like ambulatory behavior in our rat model with a RET/MEC combination (5 and 0.5 mg/kg) successfully rescued ambulation deficits, but could not be fully separated from the effect of RET alone. Further development of this approach is recommended.

Expression of TWIK-related acid sensitive K+ channels in capsaicin sensitive and insensitive cells of rat dorsal root ganglia.

Previous reports have demonstrated that small- to medium-diameter dorsal root ganglia (DRG) cells in rats can be subgrouped into individual cell types by patterns of voltage-activated currents. These cell types have consistent responses to algesic compounds and maintain characteristic histochemical phenotypes. Using immunocytochemical methods, we have now examined expression of TWIK (tandem of P domains in a weak inwardly rectifying K+ channel)-related acid sensitive K+ (TASK) channels, TASK-1, TASK-2 and TASK-3, in nine electrophysiologically identified small- to medium-diameter DRG cell types. The immunoreactivity in DRG cells was diverse, with all nine cell types expressing one to all three TASK channels. Some cells expressed TASK-1 (types 1, 4, 6 and 9), some TASK-2 (types 2, 4, 5, 6, 7 and 9), and some TASK-3 (types 1, 2, 3, 4, 5, 6 and 8). The co-expression of TASK-1 and TASK-3 in cell types 1, 4 and 6 suggests that these sensory afferents might contain functional heterodimeric channels. In peripheral sensory afferents, TASK channels have been implicated in the pain sensory transduction pathway, and can be modulated by anesthetics and neuroprotective agents. This study seeks to identify TASK channel populations in electrophysiologically characterized populations of putative nociceptive afferents.

Exposure to Gulf War Illness chemicals induces functional muscarinic receptor maladaptations in muscle nociceptors.

Chronic pain is a component of the multisymptom disease known as Gulf War Illness (GWI). There is evidence that pain symptoms could have been a consequence of prolonged and/or excessive exposure to anticholinesterases and other GW chemicals. We previously reported that rats exposed, for 8 weeks, to a mixture of anticholinesterases (pyridostigmine bromide, chlorpyrifos) and a Nav (voltage activated Na(+) channel) deactivation-inhibiting pyrethroid, permethrin, exhibited a behavior pattern that was consistent with a delayed myalgia. This myalgia-like behavior was accompanied by persistent changes to Kv (voltage activated K(+)) channel physiology in muscle nociceptors (Kv7, KDR). In the present study, we examined how exposure to the above agents altered the reactivity of Kv channels to a muscarinic receptor (mAChR) agonist (oxotremorine-M). Comparisons between muscle nociceptors harvested from vehicle and GW chemical-exposed rats revealed that mAChR suppression of Kv7 activity was enhanced in exposed rats. Yet in these same muscle nociceptors, a Stromatoxin-insensitive component of the KDR (voltage activated delayed rectifier K(+) channel) exhibited decreased sensitivity to activation of mAChR. We have previously shown that a unique mAChR-induced depolarization and burst discharge (MDBD) was exaggerated in muscle nociceptors of rats exposed to GW chemicals. We now provide evidence that both muscle and vascular nociceptors of naïve rats exhibit MDBD. Examination of the molecular basis of the MDBD in naïve animals revealed that while the mAChR depolarization was independent of Kv7, the action potential burst was modulated by Kv7 status. mAChR depolarizations were shown to be dependent, in part, on TRPA1. We argue that dysfunction of the MDBD could be a functional convergence point for maladapted ion channels and receptors consequent to exposure to GW chemicals.

Diverse immunocytochemical expression of opioid receptors in electrophysiologically defined cells of rat dorsal root ganglia.

The development of opiate analgesics that do not produce adverse side effects is hampered by the difficulty in developing drugs that are tissue/sensory cell-specific. Previously, our laboratory has demonstrated that small- and medium-diameter dorsal root ganglia (DRG) cells can be subclassified into at least nine distinct cell types based upon their patterns of voltage activated currents [Petruska, J.C., Napaporn, J., Johnson, R.D., Gu, J.G., Cooper, B.Y., 2000. Subclassified acutely dissociated cells of rat DRG: histochemistry and patterns of capsaicin-, proton-, and ATP-activated currents. J. Neurophysiol. 84 (5), 2365-2379; Petruska, J.C., Napaporn, J., Johnson, R.D., Cooper, B.Y., 2002. Chemical responsiveness and histochemical phenotype of electrophysiologically classified cells of the adult rat dorsal root ganglion. Neuroscience 115 (1), 15-30.] Based on their responses to algesic compounds and histochemical phenotype, eight of the nine subtypes are likely nociceptors. In the present study, we examined the immunoreactivity (IR) of delta-, kappa- and mu-opioid receptors (DOR, KOR and MOR, respectively), in 164 electrophysiologically subclassified DRG neurons. The expression of opioid receptors in the DRG cell types was diverse. Type 1 (25-30 microm cell diameter) and type 9 (35-45 microm) expressed MOR-IR, but were negative for DOR-IR and KOR-IR. Type 2 (25-30 microm) co-expressed DOR-IR and MOR-IR, but did not express KOR-IR. Type 3 (15-20 microm), the non-nociceptive cell type, was not immunoreactive. Type 4 (35-45 microm), type 6 (35-45 microm), and type 7 (15-20 microm) expressed all three opioid receptors. Type 5 (35-45 microm) and type 8 (35-45 microm), co-expressed KOR-IR and MOR-IR, but did not express DOR-IR. The co-expression of opioid receptors in some of the cell types suggests that these sensory afferents might contain heteromeric opioid receptors. Additionally, the diverse expression patterns of opioid receptors between cell types and the consistency of these patterns maintained within each cell type provides further evidence of distinct functional properties of DRG nociceptors.

A delayed chronic pain like condition with decreased Kv channel activity in a rat model of Gulf War Illness pain syndrome.

Following their return from deployment, Gulf War (GW) veterans reported widespread joint and muscle pain at rates that far exceeded those of soldiers returning from other conflicts. It is widely believed that exposure to insecticides, repellants and nerve gas prophylactics contributed to the symptoms of Gulf War Illness (GWI), but an animal model of GW pain has been elusive. In our previous work, we observed that 4-8 weeks exposure to pyridostigmine bromide (PB), permethrin and chlorpyrifos could produce persistent alterations in the physiology of Nav1.9 and Kv7 expressed in deep tissue nociceptors of the dorsal root ganglion. However, behavioral assessments from these same rats were not consistent with a delayed pain syndrome similar to that of GWI pain. In the present studies, we intensified the exposure to anticholinesterases PB and chlorpyrifos while retaining the same dosages. Animals receiving the intensified protocol for 30 days exhibited significant increases in resting for about 8 weeks after exposure. Thereafter, all measures were comparable to controls. Animals treated with intensified anticholinesterases for 60 days exhibited increased resting and reduced movement 12 weeks post-exposure. In whole cell patch studies, muscle and vascular nociceptor KDR and Kv7 ion channels exhibited increased amplitude relative to controls (e.g., normalized current and/or peak conductance) at 8 weeks post-exposures; however, at 12 weeks post-exposure, the amplitude of these currents was significantly decreased in muscle nociceptors. In current clamp studies, muscle nociceptors also manifested increased action potential duration, afterhyperpolarization and increased discharge to muscarinic agonists 12 weeks post-exposure. The decline in activity of muscle nociceptor KDR and Kv7 channel proteins was consistent with increased nociceptor excitability and a delayed myalgia in rats exposed to GW chemicals.

Effects of systemic morphine on responses of primates to first or second pain sensations.

Despite evidence that systemic morphine preferentially attenuates second pain sensations that are presumed to result from activation of unmyelinated (C) nociceptors, most animal models of nociception elicit sensations that result from or are dominated by activation of myelinated (A-delta) nociceptors. Therefore, methods were developed to directly compare the effects of morphine on late (second) pain sensations and early onset (first) pain sensations in an animal model. In order to establish appropriate stimulus parameters, human psychophysical experiments compared characteristics of sensations evoked by brief (pulsed) thermal stimulation and ramp-and-hold thermal stimulation. Brief (500 msec) contact of a pre-heated thermode with the skin produced late pain sensations with peripheral conduction velocities in the range of C afferents, as estimated by latencies from stimulation of proximal and distal sites on the leg. The sensations evoked by brief contact increased with successive contacts (pulses) at 0.4 Hz, demonstrating temporal summation of sensation intensity. Pretreatment of the skin with capsaicin enhanced the late pain sensations from pulsed stimulation. In contrast, peak sensations evoked by ramp-and-hold thermal stimulation were evoked at similar latencies from disparate sites on the leg, and capsaicin pretreatment of the skin did not increase the magnitude of these sensations. The pulsed and ramp-and-hold forms of stimulation were used in a paradigm designed to test for differential effects of systemic morphine on operant responses of non-human primates. Low doses of morphine reduced operant responding to pulsed thermal contact, while higher doses were required to affect responses to ramp-and-hold thermal stimulation. The low doses of morphine did not suppress non-nociceptive (intertrial) motor responses, indicating that motor inhibition was not responsible for the effects on escape responses to pulsed stimulation. Measurements of skin temperature 10 cm from the site of stimulation showed that morphine had no effect on baseline temperature but attenuated changes in skin temperature that were elicited by pulsed and by ramp-and-hold stimulation. This effect of morphine on skin temperature responses could not account for the reduction of operant responsivity to thermal stimulation. These results support previous findings that systemic morphine preferentially attenuates second pain sensations, and a new animal model of morphine-sensitive thermal nociception is established. These findings demonstrate the importance of defining the sources of afferent input and the response measures in experiments which attempt to measure antinociceptive effects of pharmacological agents.

Chemical responsiveness and histochemical phenotype of electrophysiologically classified cells of the adult rat dorsal root ganglion.

Whole cell patch recordings were obtained from medium diameter (35-45 microm) dorsal root ganglion neurons. Using electrophysiological parameters, we were able to subclassify acutely dissociated dorsal root ganglion cells into three uniform (types 5, 6 and 9) and one mixed class (type 8) of neurons. All subtypes (types 5, 6, 8 and 9) had broad action potentials (7.0+/-0.2, 5.2+/-0.4, 7.3+/-0.5 and 6.0+/-0.4 ms) and exceptionally long afterhyperpolarizations (112+/-9, 178+/-19, 124+/-31 and 204+/-33 ms). Long afterhyperpolarizations have been linked to mechanically insensitive (silent) nociceptors by other laboratories [Djouhri et al., J. Physiol. 513 (1998) 857-872]. Chemosensitivity varied among cell classes. Cell types 5, 8 and 9 were capsaicin sensitive (45+/-13, 87+/-30 and 28+/-13 pA/pF; 5 microM) groups, while the type 6 cell was capsaicin insensitive. All cell types expressed ASIC-like (acid sensing ion channel) amiloride sensitive, proton-activated currents with a threshold of pH 6.8 and a peak near pH 5.0. All medium sized cells were sensitive to ATP (50 microM) and exhibited the 'mixed' form of ATP-gated current [Burgard et al., J. Neurophysiol. 82 (1999) 1590-1598; Grubb and Evans, Eur. J. Neurosci. 11 (1999) 149-154]. Immunohistochemistry performed on individual cells indicated the expression of both P2X(1) and P2X(3) subunits. Electrophysiologically defined classes were histochemically uniform. All types were examined for the presence of substance P (SP), calcitonin gene related peptide (CGRP) and binding of isolectin B4 (IB4). All subtypes expressed CGRP immunoreactivity. Types 5 and 8 co-expressed SP and CGRP immunoreactivity and also bound IB4. Subtypes 6 and 9 were positive for neurofilament m. It is likely that these cells represent major classes of myelinated and unmyelinated peptide expressing nociceptors.

Characterization and function of TWIK-related acid sensing K+ channels in a rat nociceptive cell.

We examined the properties of a proton sensitive current in acutely dissociated, capsaicin insensitive nociceptive neurons from rat dorsal root ganglion (DRG). The current had features consistent with K(+) leak currents of the KCNK family (TASK-1, TASK-3; TWIK-related acid sensing K(+)). Acidity and alkalinity induced inward and outward shifts in the holding current accompanied by increased and decreased whole cell resistance consistent with a K(+) current. We used alkaline solutions to open the channel and examine its properties. Alkaline evoked currents (AECs; pH 10.0-10.75), reversed near the K(+) equilibrium potential (-74 mV), and were suppressed 85% in 0 mM K(+). AECs were insensitive to Cs(+) (1 mM) and anandamide (1 microM), but blocked by Ba(++) (1 mM), quinidine (100 microM) or Ruthenium Red (10 microM). This pharmacology was identical to that of rat TASK-3 and inconsistent with that of TASK-1 or TASK-2. The TASK-like AEC was not modulated by PKA (forskolin, kappa opioid agonists U69593 and GR8696, somatostatin) but was inhibited by PKC activator phorbol-12-myristate-13 acetate (PMA). When acidic solutions were used, we were able to isolate a Ba(++) and Ruthenium Red insensitive current that was inhibited by Zn(++). This Zn(++) sensitive component of the proton sensitive current was consistent with TASK-1. In current clamp studies, acidic pH produced sensitive changes in resting membrane potential but did not influence excitability (pH 7.2-6.8). In contrast, Zn(++) produced substantial changes in excitability at physiological pH. Alkaline solutions produced hyperpolarization followed by proportional burst discharges (pH 10.75-11.5) and increased excitability (at pH 7.4). In conclusion, multiple TASK currents were present in a DRG nociceptor and differentially contributed to distinct discharge mechanisms.

Distribution of P2X1, P2X2, and P2X3 receptor subunits in rat primary afferents: relation to population markers and specific cell types.

We determined the co-expression of immunoreactivity (IR) for ATP-receptor subunits (P2X1, P2X2, and P2X3), neuropeptides, neurofilament (NF), and binding of the isolectin B(4) from Griffonia simplicifolia type one (GS-I-B(4)) in adult dorsal root ganglion neurons. P2X1-IR was expressed primarily in small DRG neurons. Most P2X1-IR neurons expressed neuropeptides and/or GS-I-B(4)-binding, but lacked NF-IR. P2X1-IR overlapped with P2X3-IR, though each was also found alone. P2X2-IR was expressed in many P2X3-IR small neurons, as well as a group of medium to large neurons that lacked either P2X3-IR or GS-I-B(4)-binding. A novel visible four-channel fluorescence technique revealed a unique population of P2X2/3-IR neurons that lacked GS-I-B(4)-binding but expressed NF-IR. Co-expression of P2X1, and P2X3 in individual neurons was also demonstrated. We examined P2X subunit-IR on individual recorded neurons that had been classified by current signature in vitro. Types 1, 2, 4 5, and 7 expressed distinct patterns of P2X-IR that corresponded to patterns identified in DRG sections, and had distinct responses to ATP. Types with rapid ATP currents (types 2, 5, and 7) displayed P2X3-IR and/or P2X1-IR. Types with slow ATP currents (types 1 and 4) displayed P2X2/3-IR. Type 1 neurons also displayed P2X1-IR. This study demonstrates that the correlation between physiological responses to ATP and the expression of particular P2X receptor subunits derived from expression systems is also present in native neurons, and also suggests that novel functional subunit combinations likely exist.

P2X1 receptor subunit immunoreactivity and ATP-evoked fast currents in adult rat dorsal root ganglion neurons.

Triple fluorescent staining for P2X1 and P2X3 subunits and isolectin I-B4 (IB4) were performed on acutely dissociated rat DRG neurons. Immunoreactivity for P2X1 and P2X3 subunits was present separately or together in DRG neurons. P2X1 immunoreactivity was present in both IB4-positive and IB4-negative cells. When combining patch-clamp recordings with immunostaining for the P2X1 and P2X3 subunits on single recorded cells, ATP-evoked fast currents were shown to be present on DRG neurons that have immunoreactivity for the P2X3 subunit only, the P2X1 subunit only, or both P2X1 and P2X3 subunits. These results raised a possibility that, in addition to the P2X3 receptor subunit, the P2X1 subunit may also contribute to functional P2X receptors with fast kinetics in DRG neurons.

A stimulator for studies of mechanical nociception based upon a commercially available translation table.

We have developed a mechanical stimulator that can be used for studies of sensory physiology, psychophysics and tissue mechanics. The stimulator operates in either displacement or force-servo modes. It surpasses other available stimulators in respect to displacement range, measurement sensitivity and cost, and it can be easily assembled from parts that are commercially available.

Comparisons of dose-dependent effects of systemic morphine on flexion reflex components and operant avoidance responses of awake non-human primates.

Electromyographic activity and the force of reflex and operant responses were recorded following administration of morphine. Low doses facilitated reflex responses to input from A-delta afferents but not from A-beta input. Higher doses inhibited A-delta responses but not A-beta responses. Operant avoidance responses to visual cues were unchanged. Thus, depending on the dose, nociceptive reflexes were facilitated or inhibited, without associated effects on non-nociceptive input or on motor output.

Heat sensitization in skin and muscle nociceptors expressing distinct combinations of TRPV1 and TRPV2 protein.

Recordings were made from small and medium diameter dorsal root ganglia (DRG) neurons that expressed transient receptor potential (TRP) proteins. Physiologically characterized skin nociceptors expressed either TRPV1 (type 2) or TRPV2 (type 4) in isolation. Other nociceptors co-expressed both TRP proteins and innervated deep tissue sites (gastrocnemius muscle, distal colon; type 5, type 8) and skin (type 8). Subpopulations of myelinated (type 8) and unmyelinated (type 5) nociceptors co-expressed both TRPs. Cells that expressed TRPV1 were excellent transducers of intense heat. Proportional inward currents were obtained from a threshold of approximately 46.5 to approximately 56 degrees C. In contrast, cells expressing TRPV2 alone (52 degrees C threshold) did not reliably transduce the intensity of thermal events. Studies were undertaken to assess the capacity of skin and deep nociceptors to exhibit sensitization to repeated intense thermal stimuli [heat-heat sensitization (HHS)]. Only nociceptors that expressed TRPV2, alone or in combination with TRPV1, exhibited HHS. HHS was shown to be Ca(2+) dependent in either case. Intracellular Ca(2+) dependent pathways to HHS varied with the pattern of TRP protein expression. Cells co-expressing both TRPs modulated heat reactivity through serine/threonine phosphorylation or PLA(2)-dependent pathways. Cells expressing only TRPV2 may have relied on tyrosine kinases for HHS. We conclude that heat sensitization in deep and superficial capsaicin and capsaicin-insensitive C and Adelta nociceptors varies with the distribution of TRPV1 and TRPV2 proteins. The expression pattern of these proteins are specific to subclasses of physiologically identified C and A fiber nociceptors with highly restricted tissue targets.

Proton sensitivity Ca2+ permeability and molecular basis of acid-sensing ion channels expressed in glabrous and hairy skin afferents.

We contrasted the physiology and peripheral targets of subclassified nociceptive and nonnociceptive afferents that express acid-sensing ion channel (ASIC)-like currents. The threshold for current activation was similar in eight distinct cell subclasses regardless of functional modality (pH 6.8). When potency was determined from concentration-response curves, nonnociceptors exhibited currents with significantly greater potency than that of all but one class of nociceptors (pH50 = 6.54 and 6.75 vs. 6.20-6.34). In nonnociceptive cells, acid transduction was also confined to a very narrow range (0.1-0.3 vs. 0.8-1.4 pH units for nociceptors). Simultaneous whole cell recording and ratiometric imaging of three peptidergic nociceptive classes were consistent with the expression of Ca2+ -permeable ASICs. Sensitivity to psalmotoxin and flurbiprofen indicated the presence of Ca2+ -permeable ASIC1a. Immunocytochemistry on these subclassified populations revealed a differential distribution of five ASIC proteins consistent with Ca2+ permeability and differential kinetics of proton-gated currents (type 5: ASIC1a, 1b, 2a, 2b, 3; type 8a: ASIC1a, 1b, 3; type 8b: ASIC1a, 1b, 2a, 2b, 3). Using DiI tracing, we found that nociceptive classes had discrete peripheral targets. ASIC-expressing types 8a and 9 projected to hairy skin, but only types 8a and 13 projected to glabrous skin. Non-ASIC-expressing types 2 and 4 were present only in hairy skin. We conclude that ASIC-expressing nociceptors differ from ASIC-expressing nonnociceptors mainly by range of proton reactivity. ASIC- as well as non-ASIC-expressing nociceptors have highly distinct cutaneous targets, and only one class was consistent with the existence of a generic C polymodal nociceptor (type 8a).

Nicotinic AChR in subclassified capsaicin-sensitive and -insensitive nociceptors of the rat DRG.

Nociceptive cells of the dorsal root ganglion (DRG) were subclassified, in vitro, according to patterns of voltage-activated currents. The distribution and form of nicotinic ACh receptors (nAChRs) were determined. nAChRs were present on both capsaicin-sensitive and -insensitive nociceptors but were not universally present in unmyelinated nociceptors. In contrast, all A delta nociceptors (types 4, 6, and 9) expressed slowly decaying nAChR. Three major forms of nicotinic currents were identified. Specific agonists and antagonists were used to demonstrate the presence of alpha7 in two classes of capsaicin-sensitive, unmyelinated nociceptors (types 2 and 8). In type 2 cells, alpha7-mediated currents were found in isolation. Whereas alpha7 was co-expressed with other nAChR in type 8 cells. These were the only classes in which alpha7 was identified. Other nociceptive classes expressed slowly decaying currents with beta4 pharmacology. Based on concentration response curves formed by nicotinic agonists [ACh, nicotine, dimethyl phenyl piperazinium (DMPP), cytisine] evidence emerged of two distinct nAChR differentially expressed in type 4 (alpha3beta4) and types 5 and 8 (alpha3beta4 alpha5). Although identification could not be made with absolute certainty, patterns of potency (type 4: DMPP > cytisine > nicotine = ACh; type 5 and type 8: DMPP = cytisine > nicotine = ACh) and efficacy provided strong support for the presence of two distinct channels based on an alpha3beta4 platform. Studies conducted on one nonnociceptive class (type 3) failed to reveal any nAChR. After multiple injections of Di-I (1,1'-dilinoleyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate) into the hairy skin of the hindlimb, we identified cell types 2, 4, 6, 8, and 9 as skin nociceptors that expressed nicotinic receptors. We conclude that at least three nicotinic AChR are diversely distributed into discrete subclasses of nociceptors that innervate hairy skin.

Cutaneous texture discrimination following transection of the dorsal spinal column in monkeys.

Transection of the dorsal spinal column in monkeys has been shown to impair discrimination of the frequency or duration of repetitive tactile stimulation, without recovery over extended periods of postoperative testing. These deficits would be likely to prevent discrimination between textures presented passively and in sequence, if repetitive temporal sequences were distinguishing features of the textures. However, previous investigations of texture discrimination after dorsal column section did not obtain a deficit on tests involving active palpation of sandpaper surfaces. In the present study, rows of raised dots were stroked across the glabrous skin of one foot so that temporal entrainment of neural activity would constitute a prominent cue. The rows were oriented mediolaterally, and the textures moved proximodistally across the skin surface (varying the spacing between the rows). Four monkeys were trained to release a lever when the rougher of two textures was in contact with the skin, and the rough texture was preceded by one to three passes of a smooth texture. Stable levels of preoperative performance ranged from 78.6 to 85.7% correct responses. After interruption of the ipsilateral dorsal column, each monkey was impaired over at least 2 months of testing. One animal did not show evidence of recovery; two recovered partially from the initial deficit; and one returned to preoperative levels of performance after extensive retraining. These results are interpreted in terms of aberrant inhibitory influences which result from repetitive stimulation after a dorsal column lesion.

Effects of spinal tractotomy on spatial sequence recognition in macaques.

Five Macaca speciosa monkeys were trained to discriminate between the order of two tactile stimulus presentations (proximal-distal vs. distal-proximal) to the lateral calf. Psychophysical thresholds for tactile sequence recognition were obtained by varying the distance between the spots stimulated. Stabilized preoperative thresholds for stimulation on the lateral calf averaged 10.4 mm, which is comparable to point localization thresholds for sequential contact of the proximal limbs of humans. Complete interruption of the ipsilateral fasciculus gracilis produced elevations of thresholds within the first 3 postoperative weeks to an average of 3.6 times the control values. With training over 6 postoperative months, normal discrimination recovered. In contrast to the effects of dorsal column (DC) lesions, interruption of the lateral and ventral columns ipsilaterally or contralaterally produced no deficit or a mild and transient deficit. Monkeys with isolation of either the ipsilateral dorsolateral column (ILC) or the contralateral ventral quadrant (CVQ) exhibited substantial and enduring deficits. Thresholds were elevated an average of 5 times the control values, and recovery was not observed or was incomplete after months of training. In contrast, isolation of the ipsilateral dorsal column produced little or no threshold elevation. The results of isolating lesions complement the series of primary lesions to each cord sector, showing that spatiotactile sequence recognition for the hindlimb is served more critically by the dorsal columns than by the lateral columns.