Punctate Administration of Ficin as a human and animal model of non-histaminergic itch.

BACKGROUND: Ficin, a cysteine protease derived from fig-tree latex, has been reported to elicit itch and nociceptive sensations, though quantitative sensory studies are lacking. Cowhage containing the pruritic cysteine Mucunain, on the contrary, has been widely studied as activating polymodal nociceptors and eliciting a histamine-independent itch. OBJECTIVES: We tested whether ficin in heat-inactivated cowhage spicules would elicit itch and nociceptive sensations in humans, and analogous behaviours in mice, which are similar to those evoked by native cowhage, and whether these behaviours in mice were dose-dependent when ficin was injected intradermally. METHODS: Human volunteers rated the magnitude of itch and nociceptive sensations evoked by either native cowhage spicules or heat-inactivated spicules soaked in 1, 10 or 100 mg/mL ficin (0.03, 0.3 and 3 ng of ficin in spicule tip), applied to forearm. In mice, itch-like scratching and nociceptive-like wiping were recorded in response to either native cowhage, to heat-inactivated spicules that were either inactive or contained 100 mg/mL ficin, or to intradermal injections of 1.25, 2.5 or 5 µg/ 5 µL, each treatment applied to the cheek. RESULTS: The dose of 100 mg/mL ficin in spicules evoked comparable magnitudes of itch, nociceptive sensations and areas of cutaneous dysesthesia as native cowhage in humans and comparable itch-like scratching and pain-like wiping behaviours in mice. But to elicit similar behaviours when injected intradermally in mice a greater amount of ficin (1.25 µg) was required. CONCLUSION: Spicule delivery or intradermal injection of ficin elicits behaviours in mice that model itch and nociceptive sensations in humans, suggesting that ficin may be useful in translating mechanistic research on the neural mechanisms of pruritic and nociceptive effects of cysteine proteases between the two species.

In vivo responses of cutaneous C-mechanosensitive neurons in mouse to punctate chemical stimuli that elicit itch and nociceptive sensations in humans.

Native cowhage spicules, and heat-inactivated spicules containing histamine or capsaicin, evoke similar sensations of itch and nociceptive sensations in humans. In ongoing studies of the peripheral neural mechanisms of chemical itch and pain in the mouse, extracellular electrophysiological recordings were obtained, in vivo, from the cell bodies of mechanosensitive nociceptive neurons in response to spicule stimuli delivered to their cutaneous receptive fields (RFs) on the distal hindlimb. A total of 43 mechanosensitive, cutaneous, nociceptive neurons with axonal conduction velocities in the C-fiber range (C-nociceptors) were classified as CM if responsive to noxious mechanical stimuli, such as pinch, or CMH if responsive to noxious mechanical and heat stimuli (51°C, 5 s). The tips of native cowhage spicules, or heat-inactivated spicules containing histamine or capsaicin, were applied to the RF. Heat-inactivated spicules containing no chemical produced only a transient response occurring during insertion. Of the 43 mechanosensitive nociceptors recorded, 20 of the 25 CMHs responded to capsaicin, and of these, 13 also responded to cowhage and/or histamine. In contrast, none of the 18 CMs responded to any of the chemical stimuli. The time course of the mean discharge rate of CMHs was similar in response to each type of spicule and generally similar, although reaching a peak earlier, to the temporal profiles of itch and nociceptive sensations evoked by the same stimuli in humans. These findings are consistent with the hypothesis that the itch and nociceptive sensations evoked by these punctuate chemical stimuli are mediated at least in part by the activity of mechanoheat-sensitive C-nociceptors. In contrast, activity in mechanosensitive C-nociceptors that do not respond to heat or to pruritic chemicals is hypothesized as contributing to pain but not to itch.

Pruritic and nociceptive sensations and dysesthesias from a spicule of cowhage.

Although the trichomes (spicules) of a pod of cowhage (Mucuna pruriens) are known to evoke a histamine-independent itch that is mediated by a cysteine protease, little is known of the itch and accompanying nociceptive sensations evoked by a single spicule and the enhanced itch and pain that can occur in the surrounding skin. The tip of a single spicule applied to the forearm of 45 subjects typically evoked 1) itch accompanied by nociceptive sensations (NS) of pricking/stinging and, to a lesser extent, burning, and 2) one or more areas of cutaneous dysesthesia characterized by hyperknesis (enhanced itch to pricking) with or without alloknesis (itch to stroking) and/or hyperalgesia (enhanced pricking pain). Itch could occur in the absence of NS or one or more dysesthesias but very rarely the reverse. The peak magnitude of sensation was positively correlated for itch and NS and increased (exhibited spatial summation) as the number of spicules was increased within a spatial extent of 6 cm but not 1 cm. The areas of dysesthesia did not exhibit spatial summation. We conclude that itch evoked by a punctate chemical stimulus can co-exist with NS and cutaneous dysesthesias as may occur in clinical pruritus. However, cowhage itch was not always accompanied by NS or dysesthesia nor was a momentary change in itch necessarily accompanied by a similar change in NS or vice versa. Thus there may be separate neural coding mechanisms for itch, nociceptive sensations, and each type of dysesthesia.

MCP-1 enhances excitability of nociceptive neurons in chronically compressed dorsal root ganglia.

Previous experimental results from our laboratory demonstrated that monocyte chemoattractant protein-1 (MCP-1) depolarizes or increases the excitability of nociceptive neurons in the intact dorsal root ganglion (DRG) after a chronic compression of the DRG (CCD), an injury that upregulates neuronal expression of both MCP-1 and mRNA for its receptor CCR2. We presently explore the ionic mechanisms underlying the excitatory effects of MCP-1. MCP-1 (100 nM) was applied, after CCD, to acutely dissociated small DRG neurons with nociceptive properties. Under current clamp, the proportion of neurons depolarized was similar to that previously observed for CCD-treated neurons in the intact ganglion, although the magnitude of depolarization was greater. MCP-1 induced a decrease in rheobase by 44 +/- 10% and some cells became spontaneously active at resting potential. Action potential width at a voltage equal to 10% of the peak height was increased from 4.94 +/- 0.23 to 5.90 +/- 0.47 ms. In voltage clamp, MCP-1 induced an inward current in 27 of 50 neurons held at -60 mV, which increased with concentration over the range of 3 to 300 nM (EC(50) = 45 nM). The MCP-1-induced current was not voltage dependent and had an estimated reversal potential of -27 mV. In addition, MCP-1 inhibited a voltage-dependent, noninactivating outward current, presumably a delayed rectifier type K(+) conductance. We conclude that MCP-1 enhances excitability in CCD neurons by, at least, two mechanisms: 1) activation of a nonvoltage-dependent depolarizing current with characteristics similar to a nonselective cation conductance and 2) inhibition of a voltage-dependent outward current.

Effects of a chronic compression of the dorsal root ganglion on voltage-gated Na+ and K+ currents in cutaneous afferent neurons.

A chronic compression of the dorsal root ganglion (CCD) produces ipsilateral cutaneous hyperalgesia that is associated with an increased excitability of neuronal somata in the compressed ganglion, as evidenced by spontaneous activity and a lower rheobase. We searched for differences in the properties of voltage-gated Na+ and K+ currents between somata of CCD- and control (unoperated) rats. CCD was produced in adult rats by inserting two rods through the intervertebral foramina, one compressing the L4, and the other, the ipsilateral, L5 dorsal root ganglion (DRG). After 5-9 days, DRG somata were dissociated and placed in culture for 16-26 h. Cutaneous neurons of medium size (35-45 microm), Fluorogold-labeled from the hindpaw, were selected for whole cell patch-clamp recording of action potentials and ion currents. In comparison with control neurons, CCD neurons had steady-state activation curves for TTX-sensitive (TTX-S) Na+ currents that were shifted in the hyperpolarizing direction, and CCD neurons had enhanced TTX-resistant (TTX-R) current. CCD neurons also had smaller, fast-inactivating K+ currents (Ka) at voltages from -30 to 50 mV. The reduction in Ka, the hyperpolarizing shift in TTX-S Na+ current activation, and the enhanced TTX-R Na+ current may all contribute to the enhanced neuronal excitability and thus to the pain and hyperalgesia associated with CCD.

Inflammatory mediators enhance the excitability of chronically compressed dorsal root ganglion neurons.

A laterally herniated disk, spinal stenosis, and various degenerative or traumatic diseases of the spine can sometimes lead to a chronic compression and inflammation of the dorsal root ganglion and chronic abnormal sensations including pain. After a chronic compression of the dorsal root ganglion (CCD) in rats, the somata in the dorsal root ganglion (DRG) become hyperexcitable, and some exhibit ectopic, spontaneous activity (SA). Inflammatory mediators have a potential role in modulating the excitability of DRG neurons and therefore may contribute to the neuronal hyperexcitability after CCD. In this study, an inflammatory soup (IS) consisting of bradykinin, serotonin, prostaglandin E2, and histamine (each 10(-6) M) was applied topically to the DRG. The responses of DRG neurons were electrophysiologically recorded extracellularly from teased dorsal root fibers or intracellularly from the somata in the intact DRG or from dissociated neurons within 30 h of culture. In all three preparations, IS remarkably increased the discharge rates of SA CCD neurons and evoked discharges in more silent-CCD than control neurons. IS slightly depolarized the resting membrane potential and decreased the current and voltage thresholds of action potential in both intact and dissociated neurons, although the magnitude of depolarization or decrease in action potential threshold was not significantly different between CCD and control. IS-evoked responses were found in a proportion of neurons in each size category including those with and without nociceptive properties. Inflammatory mediators, by increasing the excitability of DRG somata, may contribute to CCD-induced neuronal hyperexcitability and to hyperalgesia and tactile allodynia.

Comparison of responses to tensile and compressive stimuli in C-mechanosensitive nociceptors in rat hairy skin.

Mechanosensitive nociceptors with unmyelinated axons (C-fibers) were studied in a preparation of isolated skin and nerve from rat. Afferent discharges were recorded while the skin was mechanically stimulated using quantitative stretch (tension) and indentation (compression). The apparatus allowed for generating stimuli of equal magnitudes in both tension and compression. Stimulus-response functions were obtained for individual afferents relating discharge rate to tensile stress or compressive stress. A response threshold and maximal slope were obtained from each function. Thresholds did not differ significantly for compression and tension nor did the maximal slopes. We conclude that C-nociceptors are equally sensitive to tensile and compressive stress.

Fine-scale organization of SI (area 3b) in the squirrel monkey revealed with intrinsic optical imaging.

Optical imaging of intrinsic cortical activity was used to study the somatotopic map and the representation of pressure, flutter, and vibration in area 3b of the squirrel monkey (Saimiri sciureus) cortex under pentothal or isoflurane anesthesia. The representation of the fingerpads in primary somatosensory cortex was investigated by stimulating the glabrous skin of distal fingerpads (D1-D5) with Teflon probes (3-mm diam) attached through an armature to force feedback-controlled torque motors. Under pentothal anesthesia, intrinsic signal maps in area 3b obtained in response to stimulation (trapezoidal indentation) of individual fingerpads showed focal activations. These activations (ranging from 0.5 to 1.0 mm) were discrete and exhibited minimal overlap between adjacent fingerpad representations. Consistent with previously published maps, a somatotopic representation of the fingerpads was observed with an orderly medial to lateral progression from the D5 to D1 fingerpads. Under isoflurane anesthesia, general topography was still maintained, but the representation of fingerpads on adjacent fingers had higher degrees of overlap than with pentothal anesthesia. Multi- and single-unit recordings in the activation zones confirmed the somatotopic maps. To examine preferential inputs from slowly adapting type I (SA) and rapidly adapting type I (RA) and type II (PC) mechanoreceptors, we applied stimuli consisting of sinusoidal indentations that produce sensations of pressure (1 Hz), flutter (30 Hz), and vibration (200 Hz). Under pentothal anesthesia, activation patterns to these different stimuli were focal and coincided on the cortex. Under isoflurane, activation zones from pressure, flutter, and vibratory stimuli differed in size and shape and often contained multiple foci, although overall topography was maintained. Subtraction and vector maps revealed cortical areas (approximate 250-microm diam) that were preferentially activated by the sensations of pressure, flutter, and vibration. Multi- and single-unit recordings aided in the interpretation of the imaging maps. In conclusion, the cortical signals observed with intrinsic signal optical imaging delineated a somatotopic organization of area 3b and revealed different topographical cortical activation patterns for pressure, flutter, and vibratory stimuli. These patterns were dependent on anesthesia type. Possible relationships of these anesthesia effects to somatosensory cortical plasticity are discussed.

Softness discrimination with a tool.

The abilities of humans to discriminate the softness of rubber objects of differing compliance with a hand-held tool (a stylus) was measured under experimental conditions that differed as to how the tool was used and the kind of sensory information available. When the subject actively tapped or pressed the compliant objects, they discriminated softness as well by means of a stylus as they did by contacting the objects directly with the fingerpad. Discrimination with the stylus was unaffected by whether the stylus was controlled by one or two fingers. While tapping or pressing a stylus held in a precision grip, the grip force increased before, reached a maximum at the same time as, and decreased in parallel with the compressional force. This relationship was suggestive of anticipatory motor control based on an internal model of the motor system and the physical properties of the object. Discrimination was significantly better when tapping as opposed to pressing the objects with the stylus. This was hypothesized as due to the presence of tactile cues generated by the rapid increase in force rate as the stylus struck and indented the object during tapping. During tapping, the magnitude and rate of compressional force produced by the stylus against the object were greater, the harder the object. An additional cue, possibly kinesthetic, during pressing and tapping was the magnitude of indentation of the specimen by the stylus that was greater, the softer the object. Subjects could discriminate differences on softness by tactile cues alone in the absence of kinesthetic when compliant objects were tapped at approximately the same velocity by the experimenter against a stylus in contact with the subject's passive fingerpad. Discrimination deteriorated if the softer specimen of a pair was tapped with a slightly greater velocity than the harder and not possible if the specimens were pressed against the stylus without generating tactile cues of mechanical contact. In contrast, discrimination was possible during active pressing and unaffected by variations in velocity during active tapping. It is concluded that during active movements, kinesthetic information and knowledge of central efferent commands provide useful cues that are not present during passive touch. These cues allow the observer to discriminate differences in object compliance not confounded by differences in applied velocity.

Mechanical and thermal hyperalgesia and ectopic neuronal discharge after chronic compression of dorsal root ganglia.

Chronic compression of the dorsal root ganglion (CCD) was produced in adult rats by implanting a stainless steel rod unilaterally into the intervertebral foramen, one rod at L(4) and another at L(5). Two additional groups of rats received either a sham surgery or an acute injury consisting of a transient compression of the ganglion. Withdrawal of the hindpaw was used as evidence of a nocifensive response to mechanical and thermal stimulation of the plantar surface. In addition, extracellular electrophysiological recordings of spontaneous discharges were obtained from dorsal root fibers of formerly compressed ganglia using an in vitro nerve-DRG-dorsal root preparation. The mean threshold force of punctate indentation and the mean threshold temperature of heating required to elicit a 50% incidence of foot withdrawal ipsilateral to the CCD were significantly lower than preoperative values throughout the 35 days of postoperative testing. The number of foot withdrawals ipsilateral to the CCD during a 20-min contact with a temperature-controlled floor was significantly increased over preoperative values throughout postoperative testing when the floor was 4 degrees C (hyperalgesia) and, to a lesser extent, when it was 30 degrees C (spontaneous pain). Stroking the foot with a cotton wisp never elicited a reflex withdrawal before surgery but did so in most rats tested ipsilateral to the CCD during the first 2 postoperative weeks. In contrast, the CCD produced no changes in responses to mechanical or thermal stimuli on the contralateral foot. The sham operation and acute injury produced no change in behavior other than slight, mechanical hyperalgesia for approximately 1 day, ipsilateral to the acute injury. Ectopic spontaneous discharges generated within the chronically compressed ganglion and, occurring in the absence of blood-borne chemicals and without an intact sympathetic nervous system, were recorded from neurons with intact, conducting, myelinated or unmyelinated peripheral nerve fibers. The incidence of spontaneously active myelinated fibers was 8.61% for CCD rats versus 0.96% for previously nonsurgical rats. We hypothesize that a chronic compression of the dorsal root ganglion after certain injuries or diseases of the spine may produce, in neurons with intact axons, abnormal ectopic discharges that originate from the ganglion and potentially contribute to low back pain, sciatica, hyperalgesia, and tactile allodynia.

Enhanced excitability of sensory neurons in rats with cutaneous hyperalgesia produced by chronic compression of the dorsal root ganglion.

Pain and hyperalgesia can occur when the dorsal root ganglion (DRG) and its roots are deformed mechanically in association with injuries or diseases of the spine. To evaluate the electrophysiological changes that contribute to this sensory pathology, intracellular recordings were obtained in vitro from DRGs that had received a chronic mechanical compression [chronic compression of DRG (CCD)]. The compression was produced by inserting L-shaped rods ipsilaterally into the intervertebral foramina, one at L(4) and the other at L(5) in rats 1-14 days before the recording. Control rats received a sham operation. Postoperatively, the threshold force applied by punctate stimulation of the plantar surface of the hind paw decreased significantly on the foot ipsilateral to the CCD (mechanical hyperalgesia) but changed little on the contralateral foot or on either foot for control rats. DRG somata were viewed through a microscope during recording and classified as small, medium, and large according to their diameters. CCD cells in each size category were more excitable than those of comparable size from control rats as judged by the significantly lowered threshold currents and action potential voltage thresholds. Spontaneous activity was recorded in 11% of all the CCD cells tested. The spontaneous activity and/or changes in both the threshold currents and action potential thresholds were observed as early as one day after injury. The association of cutaneous hyperalgesia with changes in the electrophysiological properties of DRG cells suggests a possible role for intrinsic alterations in the membrane properties of compressed DRG cells in the production and persistence of chronic pain after certain spinal injuries or pathologies of the spine.

Enhancement of experimental pruritus and mechanically evoked dysesthesiae with local anesthesia.

Pain reduces itch-a commonly known effect of scratching the skin. Experimentally produced itch from histamine is sometimes accompanied by secondary sensations of pain. The present study investigated the effects of eliminating this pain, by means of a local anesthetic, on the itch and the enhanced mechanically evoked itch and pain that occur after an intradermal injection of histamine. In ten human subjects, the volar forearm was injected with either 20 microl of 2% chloroprocaine (experimental arm), or 20 microl of saline (control arm). Histamine 10 microl was injected into each bleb, and the resulting magnitude of itch estimated. The borders of three cutaneous areas were mapped within which mechanical stimulation of the skin surrounding the bleb elicited abnormal sensations (dysesthesiae): alloknesis, defined as itch evoked by innocuous stroking, and hyperalgesia and hyperknesis, characterized, respectively, by enhanced pain and enhanced itch evoked by pricking the skin with a fine tipped filament. The magnitude and duration of itch were significantly greater and the areas of dysesthesia significantly larger for the experimental than for the control arm. It is hypothesized that there exist two classes of histamine-sensitive primary afferent neurons. One class is "pruritic", and mediates itch whereas the other is "antipruritic", and evokes a centrally mediated reduction in histamine-evoked itch and dysesthesiae. It is further suggested that the anesthetic blocked the discharges of the antipruritic afferents, preventing the central inhibition from occurring and thereby unmasking the effects of the pruritic afferents.

Attenuation of experimental pruritus and mechanically evoked dysesthesiae in an area of cutaneous allodynia.

We investigated the effects of tactile allodynia on the itch and mechanically evoked dysesthesiae produced by an intradermal injection of histamine in human volunteers. After an intradermal injection of capsaicin into the volar surface of one forearm, there developed an area of tactile allodynia to stroking and hyperalgesia to pricking the skin. Histamine was then injected simultaneously into the area of allodynia (experimental arm) and into the opposite forearm (control arm). Magnitude estimates of itch were obtained every 15 s for 5 min, and the areas of cutaneous hyperalgesia (pricking-evoked pain), alloknesis (stroking-evoked itch), hyperknesis (pricking-evoked itch) and wheal and flare were measured. The areas of wheal and flare were not significantly different on the two arms. The magnitude of itch and the areas of hyperknesis and alloknesis developed normally on the control arm but were absent or greatly reduced on the experimental arm. Thus, both the itch and the alloknesis and hyperknesis normally induced by histamine were absent or greatly reduced when histamine was injected in an area of capsaicin-induced allodynia. These results are compatible with the hypothesis that activity in capsaicin-sensitive, nociceptive primary afferent neurons evokes a central neuronal inhibitory process that prevents or reduces the itch and mechanically evoked dysesthesiae normally produced by an intradermal injection of histamine.

Raised object on a planar surface stroked across the fingerpad: responses of cutaneous mechanoreceptors to shape and orientation.

The representations of orientation and shape were studied in the responses of cutaneous mechanoreceptors to an isolated, raised object on a planar surface stroked across the fingerpad. The objects were the top portions of a sphere with a 5-mm radius, and two toroids each with a radius of 5 mm along one axis and differing radii of 1 or 3 mm along the orthogonal axis. The velocity and direction of stroking were fixed while the orientation of the object in the horizontal plane was varied. Each object was stroked along a series of laterally shifted, parallel, linear trajectories over the receptive fields of slowly adapting, type I (SA), and rapidly adapting, type I (RA) mechanoreceptive afferents innervating the fingerpad of the monkey. "Spatial event plots" (SEPs) of the occurrence of action potentials, as a function of the location of each object on the receptive field, were interpreted as the responses of a spatially distributed population of fibers. That portion of the plot evoked by the curved object (the SEPc) provided a representation of the shape and orientation of the two-dimensional outline of the object in the horizontal plane in contact with the skin. For both SAs and RAs, the major vector of the SEPc, obtained by a principal components analysis, was linearly related to the physical orientation of the major axis of each toroid. The spatial distribution of discharge rates [spatial rate surface profiles (SRSs), after plotting mean instantaneous frequency versus spatial locus within the SEPc] represented object shape in a third dimension, normal to the skin surface. The shape of the SA SRSs, well fitted by Gaussian equations, better represented object shape than that of the RA SRSs. A cross-sectional profile along the minor axis [spatial rate profile (SRP)] was approximately triangular for SAs. After normalization for differences in peak height, the falling slopes of the SA SRPs increased, and the base widths decreased with curvature of the object's minor axis. These curvature-related differences in slopes and widths were invariant with changes in object orientation. It is hypothesized that circularity in object shape is coded by the constancy of slopes of SA SRPs between peak and base and that the constancy of differences in the widths and falling slopes evoked by different raised objects encodes, respectively, the differences in their sizes and shapes regardless of differences in their orientation on the skin.

Encoding of shape and orientation of objects indented into the monkey fingerpad by populations of slowly and rapidly adapting mechanoreceptors.

The peripheral neural representation of object shape and orientation was studied by recording the responses of a spatially distributed population of rapidly and slowly adapting type I mechanoreceptors (RAs and SAs, respectively) to objects of different shapes and orientations indented at a fixed location on the fingerpad of the anesthetized monkey. The toroidal objects had a radius of 5 mm on the major axis, and 1, 3, or 5 mm on the minor axis. Each object was indented into the fingerpad for 4 s at orientations of 0, 45, 90, and 135 degrees using a contact force of 15 gwt. Estimations of the population responses (PRs) were constructed by combining the responses of 91 SA and 97 RA single afferents at discrete times during the indentation. The PR was composed of the neural discharge rates (z coordinate) plotted at x and y coordinates of the most sensitive spot of the receptive field. The shapes of the PRs were related to the shapes of the objects by fitting the PRs with Gaussian surfaces. The orientations of the PRs were determined from weighted principal component analyses. The SA PR encoded both the orientation and shape of the objects, whereas the RA PR did neither. The SA PR orientation was biased toward the long axis of the finger. The RA PR encoded orientation only for the object with the highest curvature but did so ambiguously. Only the SA PR was well fit by a Gaussian surface. The shape of the object was discriminated by the SA PR within the first 500 ms of contact, and the form of the SA PR remained constant during the subsequent 3.5 s. This was manifested by constant widths of the PR along the major and minor axes despite a peak response that decreased from its maximum at 200 ms to an asymptotic value starting at 1 s. Thus the shape and orientation of each object were coded by the shape and orientation of the SA PR.

Patch clamp recording from the intact dorsal root ganglion.

A method for patch-clamp recording from intact dorsal root ganglion (DRG) cells in rat is described. The L4 and L5 DRGs with sciatic nerve attached were excised from rats (10-15 days old) and placed in a recording chamber after removing the ganglion sheath and dissolving the connective tissue with dilute collagenase. The somata of individual cells were exposed by gentle surface cleaning through a perfusion micropipette. Somata were classified as Abeta, Adelta or C based on the cell size and the shape of the action potential (AP). Under current clamp, axonal conduction velocity (CV) was calculated from the distance between a stimulating electrode and the center of the ganglion divided by the latency of the AP elicited by stimulation of the sciatic nerve. CVs ranged from 0.2-0.8 m/s for C cells, 0.8-2.4 for Adelta and 3.2-5.0 for A/beta cells. AP threshold occurred at a significantly more positive potential in C cells than in Adelta and Abeta cells. Under voltage clamp, sodium currents were recorded from C cells. Both TTX-resistant (TTX-R) and TTX-sensitive (TTX-S currents) were demonstrated in the present study. The results demonstrate the feasibility of patch-clamp recording from intact, identified DRG cells in vitro.

Axotomy increases the excitability of dorsal root ganglion cells with unmyelinated axons.

Axotomy increases the excitability of dorsal root ganglion cells with unmyelinated axons. J. Neurophysiol. 78: 2790-2794, 1997. To better understand the neuronal mechanism of neuropathic pain, the effect of axotomy on the excitability of dorsal root ganglion (DRG) cells with unmyelinated axons (C cells) was investigated. Whole cell patch-clamp recordings were performed on intact DRG cells with intact axons or with axons transected 7-12 days earlier. C cells were identified by 1) soma size, 2) action potential morphology, 3) conduction velocity, and 4) in some cases, injection of Fast Blue into the injured nerve fibers. Axotomy reduced (more negative) action potential threshold but did not significantly change resting membrane potential, action potential duration, or maximal depolarization rate. Axotomy significantly increased the peak sodium current measured under voltage-clamp conditions. In Fast Blue-labeled (injured) cells, the tetrodotoxin (TTX)-sensitive current was enhanced while the TTX-resistant current was reduced. These results suggest that axotomy increased the excitability of C cells, possibly because of a preferential increase in expression of TTX-sensitive sodium currents.

An in vitro study of ectopic discharge generation and adrenergic sensitivity in the intact, nerve-injured rat dorsal root ganglion.

A chronic, loose constriction of the sciatic nerve in rat produces behavioral signs of spontaneous pain and cutaneous hyperalgesia (Bennett and Xie, Pain, 33 (1988) 87-107) as well as an abnormal spontaneous activity and adrenergic sensitivity of certain dorsal root ganglion (DRG) cells with axons in the injured nerve (Kajander et al., Neurosci. Lett., 138 (1992) 225-228; Xie et al., J. Neurophysiol., 73 (1995)1811-1820). The present study investigated whether the spontaneous activity and adrenergic sensitivity were intrinsic properties of injured DRG cells and manifested in vitro, i.e., not dependent on intact blood circulation and an intact, functioning sympathetic nervous system. Two weeks after a loose constriction of the sciatic nerve, the L4 or L5 DRG with its ligated nerve and dorsal root attached was removed from the rat and placed in a chamber. Extracellular recordings were made from teased dorsal root fibers. Spontaneous activity (>0.05 imp/s in 3 min) originating within or close to the DRG was often found in C-, Adelta- and Abeta-fibers from nerve-injured rats, but was rare in fibers with peripheral axons from uninjured nerve. The incidence of various patterns of spontaneous discharge was similar to that previously recorded in vivo. Nineteen of 30 C-fibers, four of five Adelta- and three of seven Abeta-fibers from injured nerve responded to different doses of norepinephrine (NE) applied topically to the DRG. Five of seven C- and one of two Abeta -fibers from injured nerve responded to clonidine, a more selective alpha2 adrenergic agonist. The thresholds ranged from 500 to 10 microM, the lowest dose delivered. None of the fibers from uninjured nerve responded to NE or clonidine (500 microM). Since the experiments were carried out in vitro in the intact DRG, the existence of spontaneous activity in DRG cells in nerve-injured rats was independent of any blood borne chemicals, such as norepinephrine. We hypothesize that abnormal activity and adrenergic sensitivity in injured DRG neurons are due to an intrinsic alteration of the cell body membrane.

Tensile and compressive responses of nociceptors in rat hairy skin.

Mechanically sensitive nociceptor afferents were studied in a preparation of isolated skin from rat leg. Each neuron was studied while the skin was subjected to tensile and compressive loading. The experiment was designed to create highly uniform states of stress in both tension and compression. Tensile loads were applied by pulling on the edges of the sample. Applied loads were used to determine the tensile stresses. Surface displacements were used to determine tensile strains. Compressive loads were applied by indenting the surface of the skin with flat indenter tips applied under force control. The skin was supported by a flat, hard substrate. Compressive stresses were determined from the applied loads and tip geometry. Compressive strains were determined from skin thickness and tip excursions. All nociceptors were activated by both tensile and compressive loading. There was no interaction between the responses to compressive and tensile stimuli (i.e., the responses were simply additive). Responses of nociceptors were better related to tensile and compressive stresses than to strains. Nociceptors responded better to tensile loading than to compressive loading. Response thresholds were lower and sensitivities were higher for tensile stress than for compressive stress. The response to compression was better related to compressive stress than to other stimulus parameters (i.e., load/circumference or simply load). Indentations of intact skin over a soft substrate such as muscle would be expected to cause widespread activation of nociceptors because of tensile stresses.

Neural encoding of shape: responses of cutaneous mechanoreceptors to a wavy surface stroked across the monkey fingerpad.

1. The role of cutaneous mechanoreceptors in the tactile perception of shape was investigated. Objects whose surfaces were shaped as a pattern of smooth, alternating convex and concave cylindrical surfaces of differing radii of curvature were constructed such that there were no discontinuities in the slope of the surface. These "wavy surfaces" were stroked across the fingerpad of the anesthetized monkey and electrophysiological responses of slowly adapting type I mechanoreceptive afferents (SAs) and rapidly adapting type I mechanoreceptive afferents (RAs) were recorded. 2. For both SAs and RAs, each convexity indenting the skin evoked a burst of impulses and each concavity of the same curvature that followed elicited a pause in response. "Spatial event plots" (SEPs) of the occurrence of action potentials as a function of the location of the object on the receptive field were obtained and interpreted as the responses of a spatially distributed population of fibers. With increasing magnitude of curvature (equivalently, decreasing radius of curvature) of convexity, the mean width of the burst in the SEPs for each fiber type (representing the width of a region of skin containing active fibers) decreased and the mean discharge rate during the burst increased. Over a range of velocities of stroking from 1 to 40 mm/s, the number of RAs activated increased with velocity, whereas SAs were active at all velocities. For both SAs and RAs, the burst rates increased with velocity, whereas the widths of the bursts and pauses remained approximately invariant. Thus the spatial measures of burst or pause width provide a robust representation of the size of a feature on the object surface. 3. For a given velocity of stroking, the spatially distributed pattern of averaged discharge rates (spatial rate profile, SRP) provided a representation of the shape of the wavy surface. The distance between neighboring peaks in the SRP for individual RAs and SAs was approximately the same as the distance between the peaks of the wavy surface. The averaged SRP for a population of SAs provided a better representation of shape than that for RAs. Whereas active regions in the SEP can be isomorphic to the two dimensional form of the stimulus "footprint" in contact with the skin surface, the SRP, which in addition encodes the features of the stimulus in the third dimension normal to the skin surface, is not isomorphic to the stimulus shape. 4. When the sizes as well as the shapes of objects are varied, it is hypothesized that a central processing mechanism extracts the invariant property of shape from the slopes of the rising and falling phases of an SRP that has been normalized for overall differences in discharge rates. These differences would be expected to occur with variations in the parameters of stimulation such as compressional force, stroke trajectory, and stroke velocity. It was shown that a common feature of the mean SRP for SAs evoked by each wavy surface convexity, regardless of its radius, was the constancy of the slope from the base to the peak and from the peak to the base. Thus a possible code for the constant curvature of a cylinder is the constancy of the slopes along the rising and declining phases of the triangular-shaped spatial response profile evoked in the SA population by the cylindrical convexity.