Brian Y. Cooper

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.

Selective Reduction of Second Pain Sensations by Systemic Morphine in Humans

&NA; A variety of forms of painful stimulation were delivered to human subjects in order to determine whether therapeutic dosages of systemic morphine might produce significant attenuation of some forms of phasic pain that are tolerable for experimental usage. Consistent with previous reports, simple application of thermal or electrical energy to the skin (for 3 sec) produced sensations of pain that were not significantly reduced by prior administration of morphine. Similarly, subjects that were trained to focus their attention on the magnitude of the immediate (first) pain sensation evoked by brief electrical or mechanical stimulation did not report reduction by morphine of pain attributed to conduction in myelinated peripheral nociceptors. In contrast, the magnitude of late (second) pain sensations produced by brief pulses of electrical, thermal or mechanical stimuli to the same subjects was consistently reduced significantly by doses of 5 or 10 mg of morphine. The simplest interpretation of the effect on second pain intensity is that morphine preferentially attenuates input from unmyelinated nociceptors. This conclusion was reinforced by an experiment in which chemicals were applied to the skin. Morphine reduced pain produced by capsaicin (presumed to selectively excite unmyelinated peripheral afferents) but did not diminish pain elicited by bradykinin (presumed to excite A&dgr; and C nociceptors). Comparing long duration pains from chemical stimulation (lasting in excess of 5 min) with briefer pains elicited by 50 msec to 3 sec of stimulation did not support the notion that morphine acts selectively on tonic pain. Also, after‐sensations that could be discerned following second pain were not eliminated by morphine, and paired pulse facilitation of first pain sensations remained after administration of morphine, indicating that temporal summation is not preferentially reduced. Regardless of duration, frequency or latency, pain arising exclusively from unmyelinated nociceptors was attenuated substantially, but other elicited sensations were not reliably affected. For example, detection thresholds for warmth were unaffected by morphine, demonstrating that input from all unmvelinated afferents is not reduced.

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.

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

&NA; 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.

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.

Measurement of pain and morphine hypalgesia in monkeys.

&NA; In order to determine the relative sensitivities of different behaviors to systemic morphine, monkeys were trained: (a) to escape electrical stimulation (ES) at intensities that defined escape thresholds and permitted quantification of reactions to sub‐ and suprathreshold stimuli, (b) to perform the same operant response to auditory stimulation for food reinforcement, and (c) to detect minimal intensities of light tactile stimulation, defining thresholds for touch. Thresholds for escape responses corresponded to pain thresholds of human subjects previously tested with identical stimulus parameters. The response measures that best differentiated suprathreshold levels of stimulation of the hind limbs were the force and the speed of escape responses by the forelimbs. Reflexive responses of the stimulated leg were related to ES intensity by a negatively accelerating function that was flat through much of the range of stimulus intensities that were escaped. Frequency histograms of adjunctive behaviors in the intertrial intervals revealed little, if any, relationship to the presence or the intensity of ES. The frequencies of intertrial vocalizations, spontaneous bar pulls and general bodily activity were similarly distributed following subthreshold vs. suprathreshold levels of ES and following ES vs. food reinforced trials. Dose‐response curves for the different behavioral measures revealed significant effects of systemic morphine at the following dosages: (a) The adjunctive behaviors clearly were the most susceptible to depression. Intertriai vocalizations, bar pulls and activity were reduced significantly in frequency at 0.25 mg/kg and above, (b) At doses of 1 mg/kg and above, the percentage, speed and force of escape responses were reduced. The effects on response tendency and latency cannot be ascribed with confidence to an inhibition of pain, since the percentage and speed of responses for food reinforcement also were reduced at these dosages; and thresholds for detection of light touch were significantly elevated. The force of appetitive responses was not significantly reduced by moderate doses of morphine, suggesting that reduction of escape force may represent an effect on pain sensitivity, (c) The magnitudes of reflexive responses to ES were increased by doses below 3 mg/kg, and 3–5 mg/kg attenuated reflexive force. The low doses appeared to release an inhibition of reflexive vigor that could be demonstrated by behavioral disruption prior to ES. At the doses that depressed reflex reactions, the animals gave evidence of a powerful behavioral suppression that virtually eliminated adjunctive responses and reduced the frequency, speed and vigor of operant responses to non‐nociceptive or nociceptive stimulation. These results document a requirement of high levels of systemic morphine to attenuate reactions to phasic pain from activation of myelinated peripheral afferents. At the doses that are commonly used to evaluate reactions of laboratory animals to somatosensory stimuli, rigorous control procedures are required to factor out generalized behavioral suppression.

Altered Precision Grasping in Stumptail Macaques after Fasciculus Cuneatus Lesions

Patterns of precision grasp are described in stumptail macaques (Macaca arctoides) before and after lesions of the fasciculus cuneatus (FC). Three monkeys were videotaped while reaching for and grasping small food items. From these videotapes, records were made of the style and outcome of each grasp. Kinematic measurements were also made to describe grip formation and terminal grasp. During grip formation, grip aperture was measured as the distance between the tips of the index finger and the thumb. For terminal grasp, the joint angles of the index finger were measured. The majority of grasps by normal monkeys were of the precision type, in which the item was carried between the tips of the index finger and thumb. Each normal monkey approached objects with a highly consistent grip formation; that is, the fingertips formed a small grip aperture during the approach, and the aperture varied little on repeated grasps. To grasp an item, the forefinger moved in a multiarticular pattern, in which the proximal joint flexed and the distal joint extended. As a result of this combination of movements, the forefinger pad was placed directly onto the object. Following FC transection, the monkeys were studied for 10 months, beginning 1 month after the lesion, to allow for recovery from the acute effects of surgery. The monkeys could grasp the food items, but they rarely opposed the fingertips in precision grasp. Grip formation was altered and was characterized either by excessive grip aperture or by little to no finger opening. All of the monkeys used the table surface to help grasp items. Combined multiarticular patterns of flexion and extension were never observed postoperatively; they were replaced by flexion at all joints of the fingers. These results suggest that the FCs are more important for precision grasping than for other, less refined grasp forms (e.g., power grasps; Napier, 1956). The FCs provide critical proprioceptive feedback to cerebral areas involved in the planning and/or the execution of these movements.

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.

Sensory nerve endings in the hard palate and papilla incisiva of the goat

Abstract The sensory innervation of the papilla incisiva in the hard palate of the domestic goat was studied with light and electron microscopy, supplemented by electrophysiological studies of free nerve endings. The goat lacks incisor teeth. Grass and leaves are not bitten, but pulled off by pressing them between the tongue and papilla incisiva. Thus, the masticatory mucosa is subject to particularly heavy mechanical loads requiring functional specialization of the horny epithelium in the form of thickening, i.e., the papilla incisiva and 12–14 pairs of rugae palatinae. A thin layer of firm connective tissue (lamina propria) attaches the mucosa to the periost of the hard palate. Sensory nerve fibers were found most abundantly in the papilla incisiva. Their number decreased drastically in aboral direction. A section through the first four rugae palatinae contains only about 10% of the number of free nerve endings found in the same area of mucosa from the papilla incisiva. Four types of sensory nerve endings were found. Free nerve endings were seen ubiquitously in the epithelium and superficial layer of the lamina propria. Merkel nerve endings were found in the bases of the epithelial thickenings in the papilla incisiva and rugae palatinae. Few Ruffini corpuscles were found in the deeper layer of the lamina propria, while lamellated corpuscles were seen just below the basement membrane of the epithelial pegs. Thus, a variety of sensory nerve endings were found in the hard palate, especially in those areas that are in close contact with the tongue during chewing of food. This rich innervation suggests an important role in monitoring the mechanical properties of food. Recordings were made from cell bodies supplying these terminals. Classic low-threshold, slowly adapting responses were observed in Aß afferent populations. This activity was probably mediated by Merkel type endings. Alternately, high-threshold and suprathreshold responses obtained from Aδ category afferents were likely to be nociceptive. In support of this, threshold and suprathreshold sensitization was observed following injection of serotonin into the receptive field of Aδ populations. This activity was likely to be derived from the aforementioned free nerve endings.

Resiniferatoxin-induced loss of plasma membrane in vanilloid receptor expressing cells.

Resiniferatoxin (RTX), a potent analog of capsaicin, was evaluated electrophysiologically in dorsal root ganglion (DRG) cells and cell lines ectopically expressing the vanilloid receptor type 1 (VR1) to determine if cell phenotype influenced RTXs neurotoxic properties. Furthermore, capsaicin and heat activation of VR1 were evaluated in these cells to determine if cellular damage was unique to RTX activation of the receptors. RTX application to DRG cells identified as type 1, 2 or 5, cell types known to express VR1, induced large inward currents. RTX did not induce currents in DRG cells that do not express the receptor (type 4 cells). In cell lines ectopically expressing VR1, RTX-induced similar currents. RTX produced no effect in non-transfected cells. After exposure to RTX both DRG cells and transfected cells failed to respond to subsequent applications of the agonist. In addition, whole cell capacitance was reduced up to 70%. The decrease in capacitance was associated with the loss of plasma membrane, as determined by confocal microscopy. Cell phenotype, other than VR1 expression, did not influence the response to RTX. Interestingly, capsaicin and heat activation of vanilloid receptors also decreased cell capacitance, but the loss of membrane was not as great as with RTX and responses to these stimuli were not lost after the initial exposure. The loss of cell membrane required elevated intracellular levels of Ca2+. From these data it was concluded that the loss of cell membrane was dependent on the presence of both VR1 and intracellular Ca2+ accumulation, but not on cell phenotype.