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The Journal of Neurophysiology Vol. 87 No. 3 March 2002, pp. 1280-1289
Copyright ©2002 by the American Physiological Society
Section of Neurobiology, Physiology and Behavior, University of California, Davis, California 95616
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Jinks, Steven L. and E. Carstens. Responses of Superficial Dorsal Horn Neurons to Intradermal Serotonin and Other Irritants: Comparison With Scratching Behavior. J. Neurophysiol. 87: 1280-1289, 2002. Scratching behavior is used to assess itch sensation in animals, but few studies have addressed the relative scratch-inducing capacity of different algesic and pruritic chemicals. Furthermore, central neural mechanisms underlying itch are not well understood. We used electrophysiological and behavioral methods to investigate the ability of several irritant chemicals to excite neurons in the superficial dorsal horn, as well as to elicit scratching, in rats. In anesthetized rats, single neurons in the superficial lumbar dorsal horn, identified by their responsiveness to intracutaneous (ic) histamine, were classified as wide dynamic range (WDR) or nociceptive-specific (NS). Serotonin (5-HT) given ic to the paw excited most (88%) WDR and NS neurons over a prolonged time course (often up to 40 min). 5-HT-evoked responses exhibited significant tachyphylaxis. Most neurons also gave shorter-duration responses to ic capsaicin (92%) and mustard oil (71%). In separate behavioral experiments, significant dose-related hind limb scratching directed at the ic injection site in the back of the neck was elicited by 5-HT over a time course similar to that of evoked neuronal firing. A second 5-HT injection made 40 min later at the same site elicited significantly less scratching. Formalin also elicited scratching that was not dose-related and less than that evoked by 5-HT. 5-HT and Formalin also evoked head or whole-body shakes that were significantly correlated with scratching. Neither histamine, capsaicin, nor vehicle controls elicited significant scratching or shaking. In rats, 5-HT appears to be more pruritogenic than histamine as assessed by scratching and shaking behavior, and excites superficial dorsal horn neurons over a behaviorally relevant time course. However, because most neurons additionally responded to pain-producing stimuli, they are not itch-specific. They might nonetheless contribute to neural pathways that distinguish between pain and itch based on some neural mechanism such as frequency coding.
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Itching (pruritus) is
normally evoked by insect bites or contact with nettles, but chronic
itch is associated with dermatitis and kidney or liver disease and
often is only poorly controlled. There is a general lack of knowledge
about neural itch mechanisms. We recently reported that neurons in both
superficial and deep laminae of the lumbar dorsal horn responded to
histamine, which is pruritic in humans (Simone et al.
1987
, 1991), as well as to algesic chemical
(capsaicin, mustard oil, nicotine) and physical stimuli
(Carstens 1997; Jinks and Carstens 1999,
2000a). The present study sought to further investigate
neural itch mechanisms.
The association of itch with the desire to scratch provides a rationale
to use scratching behavior to assess itch in animals. Many pruritogens,
including histamine, serotonin (5-HT), substance P, leukotriene B4, and
platelet activating factor, elicit dose-related scratching
(Andoh and Kuraishi 1998; Andoh et al.
1998; Berendsen and Broekkamp 1991;
Kitagawa et al. 1997; Kuraishi et al.
1995; Woodward et al. 1995; Yamaguchi et
al. 1999). Recent studies indicate that 5-HT is a much more
potent inducer of scratching in mice compared with histamine
(Kuraishi et al. 1995; Yamaguchi et al. 1999). Algesic chemicals such as capsaicin elicited little if any scratching in mice (Kuraishi et al. 1995) or rats
(Frenk et al. 1988), suggesting that scratching behavior
differentiates between pruritic versus algesic chemical stimuli.
The present study had two main aims. First, we investigated if
intracutaneous (ic) 5-HT excites superficial dorsal horn neurons. Second, in correlative behavioral experiments we have investigated whether pruritic (5-HT, histamine) or algesic chemicals (capsaicin, Formalin) induce scratching behavior in the rat. We hypothesized that
superficial dorsal horn neurons involved in signaling itch should be
excited by pruritic but not algesic chemcals over a time course
matching that of scratching behavior. An abstract of this work has
appeared (Jinks and Carstens 2000b).
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METHODS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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All experiments were approved by the UC Davis Animal Use and Care Advisory Committee. Adult male Sprague-Dawley rats, weighing 450-580 g, were used. Rats had continuous access to food and water and were kept on a 12-h light-dark cycle with lights on at 8 AM.
Electrophysiological experiments
Experiments were conducted using 14 rats, 4 of which had been
previously tested for scratching behavior (see following text). Rats
were anesthetized with pentobarbital sodium (induction: 65 mg/kg ip,
and maintenance: 10-20 mg · kg
1
· h1 iv), and the lumbar spinal cord was
exposed by laminectomy to record extracellular single-unit activity.
Chemicals were delivered ic in 1-µl volumes to skin in the receptive
field of the dorsal horn unit via a 30.5-gauge needle (Carstens
1997; Jinks and Carstens 2000a).
An ic histamine search strategy (Jinks and Carstens
2000a) was used to identify superficial dorsal horn units.
Immediately after ic histamine, the recording microelectrode searched
the superficial dorsal horn (down to 300 µm) for an actively firing unit. All presently recorded units were isolated <2 min after delivering the first histamine search stimulus (n = 17 of 24) or after a second search stimulus delivered at the same
(n = 3) or different (n = 4) skin site.
Once a unit was isolated, we waited for the firing rate to decline to a
steady baseline (usually 10-12 min) and then reinjected histamine at
the same site. Figure 2, A and D, shows examples
of this procedure.
When unit activity once again returned to a steady baseline, responsiveness to low-threshold mechanical stimulation was tested, and receptive fields were provisionally mapped with a weak von Frey filament (1.2-g bending force). Unit responses to other stimuli were then tested in the following order: 0.9% NaCl ic, 5-HT ic (1%), heat delivered by a 1-cm2 Peltier thermode (52°C, 5 s), noxious mechanical stimulation, ic capsaicin (0.03%), and topical application of mustard oil (10%; Fluka). This ensured that unit responses to the potential pruritogens, histamine and 5-HT, were not depressed by prior noxious stimuli. Only after applying 5-HT was the unit's mechanical responsiveness more thoroughly assessed. Units were classified as wide dynamic range (WDR) if they responded to innocuous levels of mechanical stimuli as well as to noxious mechanical (pressure, pinch) and heat stimuli. They were classified as nociceptive-specific (NS) if they did not respond to innocuous mechanical stimuli but did respond to noxious levels of mechanical pressure-pinch, and to noxious thermal stimuli. One unit did not respond to noxious mechanical stimuli and was classified as mechanically insensitive. A response was considered positive if the stimulus elicited a >200% increase in firing rate above the prestimulus level.
In six rats, only one unit was recorded. In eight rats, two units were
recorded [simultaneously in 2; 1 on each side of the cord in 6; on the
same side but at spatially separate (heel vs. toe) locations in 2]. In
the two dual-unit recordings, action potential waveforms were
discriminated using a template-matching procedure (Forster and
Handwerker 1990).
At the conclusion of the experiment, the recording site was lesioned electrolytically, and spinal cords were removed and fixed in 10% Formalin. Later, 50-µM sections of spinal cord, counter-stained with neutral red, were examined by light microscopy to identify lesion sites.
Peak firing rates evoked by ic chemical or heat stimuli were calculated by constructing peristimulus time histograms (PSTHs, binwidth: 1 s) and selecting the maximum value. Latency to peak firing rate was measured from the time of injection. Tachyphylaxis was assessed by comparing the mean total number of impulses recorded during the 15-min period following the initial and a second ic injection of 5-HT (made 30 min later) using a paired t-test, with significance occurring at a P value <0.05. For each chemical tested, significant increases in firing rate were assessed at different time intervals postinjection by averaging the firing rate over this time interval for all units, and comparing mean firing rate with mean preinjection spontaneous firing rate using a two-factor ANOVA, followed by post hoc, paired t-tests, with significance occurring at a P value <0.05. Averaged PSTHs of responses of WDR and NS units are displayed separately (Fig. 1). However, since there were not obvious differences between these groups, they were pooled for purposes of data analysis.
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Scratching behavior
These experiments quantified the number of bouts of hindlimb scratching directed at a site of irritant chemical injection into the nape of the neck. All rats were habituated to a loose cylindrical Plexiglas restrainer in four 30-min sessions the week prior to conducting experiments. At least 3 days prior to receiving injections, rats were briefly anesthetized with 3% halothane, and the fur over the rostral portion of their back was removed. The following irritant chemicals were tested: 5-HT (0.5, 1, or 2%; Sigma Chemical, St. Louis, MO; n = 8 rats tested at all 3 concentrations); Formalin (2 and 5%; n = 7 rats tested at both concentrations), capsaicin (0.01, 0.03, or 0.1%; Sigma; n = 5 rats tested at all 3 concentrations), and histamine (3 or 10%; Sigma; n = 4 rats tested at both concentrations), and were dissolved in 0.9% sodium chloride, except for capsaicin, which was dissolved in 40% ethanol at a 0.1% concentration, and then diluted with 0.9% NaCl as necessary. Vehicles (0.9% NaCl; n = 4 rats previously tested with 5-HT; and 40% ethanol, n = 5 rats previously tested with capsaicin) were also tested.
Rats were placed in the recording chamber for 30 min before receiving the chemical injection. Rats received consecutive injections at least 3 days apart in mixed order. All ic injections were given ic into the skin on the nape of the neck in a 10-µl volume. Microinjections were made by placing the rat into the restrainer for 5 min, and then gently grasping skin of the rostral back with rubber-coated forceps. A 30.5-gauge needle, connected to a 50-µl hamilton microsyringe via PE-50 tubing, was inserted into the superficial skin and left in place for 5 min, at which time the chemical was injected over a 20-s time period. The needle was left in place for 10 s postinjection, removed, and each rat was then immediately placed into a separate 30 cm × 30 cm × 40 cm opaque, open-top, plastic chamber, and videotaped from above for 40 min.
To test for tachyphylaxis, a separate group of rats (n = 7) received a microinjection of 2% 5-HT as described, videotaped for 40 min, and then immediately reinjected at the same site (marked by felt-tip pen) with the same dose of 5-HT and videotaped for another 40 min.
Four rats were injected and videotaped at a time. The experimenter left the room shortly after the injections were made and rats placed in the chamber. Videotapes were later analyzed for the number of hindlimb scratching bouts, which were collected in 2-min time bins over the 40-min videotaping period. A series of one or more scratching movements directed at the injection site was defined as a scratching bout, which ended when the rat either licked its hind paw or placed its hind paw back on the floor. The number of episodes of head or whole-body ("wet dog") shakes/40 min was also counted. The investigators scoring videotapes were blinded as to which treatment the rats had received.
Comparisons of numbers of scratching bouts and head/whole-body shakes between each treatment group and vehicle controls, as well as between different doses of a given chemical, were made using a one-factor ANOVA, followed by post hoc t-tests, with significance occurring at a P value <0.05.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Electrophysiology
UNIT SAMPLE. Eighteen WDR and six NS units (1 of which was mechanically insensitive) were recorded. The mean depth of recording was 142 ± 113 (SD) µM, and histologically recovered recording sites were located in superficial laminae of the dorsal horn (Fig. 1). For WDR units, the low-threshold mechanosensitive receptive fields spanned more than (n = 3) or less than (n = 15) 50% of the plantar surface, in the region of the toes (n = 11), mid-paw (n = 4), or heel (n = 3).
When first isolated, all units displayed a moderate to high firing rate (presumably from the histamine search stimulus), which decayed to a baseline level, usually within 12 min. All 24 units responded to a subsequent histamine injection. Figure 2, A and C, shows examples of the initial firing and response to the second histamine stimulus. During 30 s immediately preceding the histamine-evoked response, the mean spontaneous firing rate was 2.8 ± 3.4 Hz (0-3 Hz in 67%, 3-6 Hz in 25%, and 6-10 Hz in 7%). The mean peak response to the histamine injection was 69 ± 60 impulses/s, occurring at latency of 8.2 ± 9.2 s. Within 7-9 min posthistamine, the mean firing rate was not significantly different from preinjection firing (P = 0.21, paired t-test).
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VEHICLE CONTROL. The effect of vehicle injection (0.9% NaCl) was assessed in 15 units (12 WDR and 3 NS; Fig. 1, A and B, far-right PSTHs). For all units pooled, NaCl elicited a small but significant increase in firing rate during the first minute postinjection (P < 0.02, paired t-test), which was no longer significant at 1.5-2.5 min postinjection.
RESPONSE TO 5-HT AND TACHYPHYLAXIS. Immediately preceding the first 5-HT injection, the mean spontaneous firing rate was 2.9 ± 3.6 Hz, which was not significantly different from the prehistamine level (P = 0.59; paired t-test). 5-HT 1% ic elicited prolonged responses in 21/24 (88%) units (WDR: 15/18; NS: 6/6; Table 1) lasting at least 15 min, and in 10 units at least 25 min. 5-HT usually elicited a bi- or multiphasic response pattern; the averaged responses of WDR and NS units are shown in Fig. 1, A and B, respectively, and individual examples are shown in Fig. 2. The initial phasic component seen in all but one unit (Fig. 2A) had a peak firing rate (41 ± 24 impulses/s) that occurred at a mean latency of 14.2 ± 11.2 s, and then declined rapidly. This was followed by a gradual increase in firing rate that peaked at 8.2 ± 3.2 min. The mean peak firing rate of this second phase (28 ± 15 impulses/s) was significantly lower than the initial phasic response, but was significantly higher than the preinjection spontaneous firing rate (P < 0.0001, paired t-test). The mean firing rate was still significantly greater than mean spontaneous firing at 15-17 min post-5-HT (P < 0.001, paired t-test). Unit responses and scratching behavior elicited by ic injection of 5-HT 1% both exhibited prolonged time courses (compare Fig. 5A with Fig. 1A and B, 2nd PSTHs from left).
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RESPONSES TO ALGESIC STIMULI. Units were additionally tested for their response to noxious heat, ic capsaicin, and topical mustard oil. Table 1 summarizes the responses. Of the NS units, 100% responded to capsaicin and mustard oil, and 67% to noxious heat. Of the WDR units, 88% responded to capsaicin, 56% to mustard oil, and 73% to heat. Examples of responses to different stimuli are shown for a mechanically insensitive NS unit in Fig. 2A, for WDR units in Figs. 2, B and D, and 3B, and for an NS unit in Fig. 2C.
Capsaicin characteristically elicited a short-latency increase in firing rate that peaked (64 ± 42 impulses/s) at 13.4 ± 11.3 s and then decayed within 7-9 min to a rate that was not significantly different from the preinjection level (P = 0.74, paired t-test). Figure 1, A and B, shows averaged responses to capsaicin of WDR and NS units, respectively. Responses to mustard oil usually consisted of a gradual increase in firing rate over 1-2 min followed by decay to baseline within 10 min. Averaged responses to mustard oil are also shown in Fig. 1. There were very few instances in which histamine- and 5-HT-responsive units did not respond to capsaicin or mustard oil (Table 1). An example of a WDR unit that responded to histamine and 5-HT, but not to capsaicin or mustard oil (and minimally to heat) is shown in Fig. 2D. This unit's ongoing activity was higher during the capsaicin and mustard oil trials (3rd and 4th PSTHs from left), compared with posthistamine (1st PSTH), possibly due to prior 5-HT. However, neither capsaicin nor mustard oil elicited any further increase in firing.Scratching and shaking behavior
5-HT. Vehicle (NaCl 0.9% or ethanol 40%) evoked almost no scratching (Fig. 4A). 5-HT reliably induced significant hindlimb scratching in each of eight rats (P < 0.01, 1-factor ANOVA) in a concentration-dependent manner (P < 0.01, paired t-test; Figs. 4A and 5A). 5-HT also elicited a significantly greater number of "wet dog" shakes above vehicle control (P < 0.02, 1-factor ANOVA; Fig. 4A). Indeed, there was a significant correlation (r = 0.72; P < 0.001) between the total number of scratching bouts and shakes recorded per animal per session under all treatment conditions (Fig. 4D). Mean 5-HT-evoked scratching behavior began after a 2- to 4-min latency and exhibited a bimodal distribution, with the first peak at 10-12 min and a later peak at 28-30 min (Fig. 5A).
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CAPSAICIN. Capsaicin elicited low to moderate scratching and shaking behavior in some rats (44% of trials), but overall this was not significantly different from controls, nor was it dose-related (Fig. 4B), and was usually less vigorous and of shorter duration compared with 5-HT and Formalin-induced scratching.
FORMALIN. Formalin evoked low to moderate scratching that was consistent (7/7 rats) and significantly greater than vehicle (P < 0.02, 1-factor ANOVA), but was not concentration-related at the concentrations tested (Fig. 4C, left filled bars). Mean Formalin-evoked scratching behavior began at a latency of 8-10 min and peaked at 14-16 min (Fig. 5B). Formalin also evoked a significantly greater number of "wet dog" shakes compared with vehicle control (P < 0.02, 1-factor ANOVA; Fig. 4C, left open bars).
HISTAMINE. Histamine at the concentrations tested did not cause significant scratching or shaking behavior (Fig. 4C, right bars).
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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We presently identified a population of histamine-sensitive neurons in the superficial dorsal horn that additionally responded to ic 5-HT over a time course that matched scratching behavior. Tachyphylaxis was observed in the behavioral and neuronal responses to repeated injection of 5-HT. These correlations suggest that the 5-HT-responsive neurons may be involved in signaling itch, as discussed further below.
Neurobehavioral correlates and methodological considerations
We presently used a search strategy designed to identify neurons
responsive to ic histamine. This strategy could identify potential
histamine-selective neurons, but has the drawback that the overall
proportion of histamine-responsive WDR and NS neurons in the
superficial dorsal horn cannot be specified. Using a mechanical search
strategy, we found that a large majority of WDR neurons in the deep
dorsal horn responded to ic histamine and other irritants (Carstens 1997; Jinks and Carstens
1999). Using antidromic stimulation to identify spinothalamic
tract neurons in cat, Andrew and Craig (2001) recently
identified a small subpopulation of mechanically insensitive,
histamine-responsive lamina I neurons, some of which also responded to
mustard oil. Thus while our results indicate that a majority of WDR and
NS neurons in superficial dorsal horn respond nonselectively to
different irritants, a subset may have more restricted chemoselectivity
and thus be well-suited to selectively signal itch.
After initiating the electrophysiological studies, we observed that
histamine did not elicit scratching or shaking behavior and thus may
not be pruritic in Sprague-Dawley rats. Instead, 5-HT was found to
elicit dose-related scratching as well as neuronal firing over a
behaviorally relevant time course. An electrophysiological strategy
using ic 5-HT might be more appropriate to search for itch-related
dorsal horn neurons in rats. However, a drawback is the partial
tachyphylaxis to repeated ic 5-HT (Fig. 3). While we showed previously
that dorsal horn neuronal responses to histamine do not exhibit
significant tachyphylaxis or sensitization (Carstens 1997; Jinks and Carstens 2000a),
cross-tachyphylaxis induced by 5-HT might lower the incidence of
neurons responsive to other subsequently delivered chemical stimuli.
However, such an effect is not absolute, given the present finding that
a majority of units responded to capsaicin and mustard oil
following application of 5-HT (Table 1).
Recent psychophysical studies show that histamine elicits significantly
stronger and more prolonged itch when injected into a skin "bleb"
previously injected with local anesthetic (Atanassoff et al.
1999). It was hypothesized that histamine excites both "antipruritic" and itch-signaling primary afferents; the former engage a spinal process to inhibit itch transmission and are more strongly suppressed by the local anesthetic. In this scenario, it
cannot be determined whether pruritogen-responsive neurons signal itch
or are antipruritic (e.g., inhibitory interneurons). While it is
advantageous to identify ascending projections (Andrew and Craig
2001), it is virtually impossible to determine whether a
nonprojecting neuron functions as an excitatory or inhibitory interneuron. We presently made no attempt to identify the projections of the recorded neurons, which might represent a functionally heterogeneous population.
A methodological disparity is that different skin areas were injected
in the behavioral (rostral back) and electrophysiological (hind paw)
experiments. We chose to perform electrophysiological studies with
lumbar neurons for comparison with prior studies (Carstens
1997; Jinks and Carstens 1998,
1999, 2000a), while assessing hindlimb
scratching directed toward the back because this has been used in most
prior behavioral studies (e.g., Kuraishi et al. 1995).
It would be ideal to compare behavioral and neural responses to
stimulation at the same skin site. However, it is problematic to record
from neurons with input from the upper back because their receptive
fields are in the surgical field. A fruitful approach would be to
assess behavioral reactions to chemical injections into the hind paw.
Hindpaw injection of Formalin (Dubuisson and Dennis
1977) or capsaicin (Gilchrist et al. 1996)
elicits reactions (lifting, limb-guarding, licking, etc.) suggestive of
pain and hyperalgesia. Hind paw injection of 5-HT induces hyperalgesia (Abbott et al. 1996; Sufka et al. 1992;
Tokunaga et al. 1998) and was recently reported to
elicit relatively more biting and less licking of the paw compared with
Formalin (Hagiwara et al. 1999), prompting the authors
to suggest that biting may reflect itch. More information on behavioral
patterns elicited by pruritic and algesic chemical stimulation of the
paw would be useful in developing animal models that distinguish itch
from pain sensation. A drawback of our present approach was the absence
of obvious signs of pain following ic injection of algogens into the
back skin, although we noted informally that some rats vocalized at the
time of injection of histamine and capsaicin.
Another methodological disparity was our use of different ic injection
volumes in the behavioral (10 µl) and electrophysiological (1 µl)
experiments. The 1-µl injection volume was used in the electrophysiological experiments to allow comparison with our prior
studies (Carstens 1997; Jinks and Carstens
1999, 2000a) and to reduce edema and to
facilitate clearance from the dermis, thereby allowing multiple ic
injections at the same site. The 10-µl volume was used in the
behavior study because pilot studies revealed that injection of 1 µl
of 2% 5-HT in the back did not elicit scratching even though it
excited dorsal horn neurons. This difference might reflect the
requirement of spatial summation of chemonociceptive neurons to trigger
scratching behavior.
ROLE OF SUPERFICIAL DORSAL HORN NEURONS IN ITCH AND PAIN.
Several theories have been proposed for the neural encoding of itch and
pain sensations. "Specificity" theory holds that itch and pain are
signaled separately by distinct pathways. Variants of the specificity
theory incorporate the fact that pain inhibits itch by occlusion
(Handwerker 1992) or central inhibitory
mechanisms (Atanassoff et al. 1999; Brull et al.
1999; McMahon and Koltzenburg 1992). The
alternative "intensity" theory states that a common pathway signals
both itch and pain, and that these qualities are distinguished based on
some neural code such as firing frequency (McMahon and
Koltzenburg 1992; von Frey 1922).
; Jinks and Carstens 1999). While this is at odds with specificity, it is consistent with intensity theory. It is noteworthy that neuronal responses to 5-HT were of lower frequency but longer duration compared
with responses to histamine, capsaicin, or mustard oil (Fig. 1).
Furthermore, the time course of 5-HT-induced neuronal firing was
comparable to that of scratching, and both neuronal and behavioral
responses to 5-HT exhibited tachyphylaxis (Figs. 3 and 6). These
correlations support a role for 5-HT-responsive neurons in signaling
itch. Within the context of intensity theory, low-frequency firing of
such neurons may provide a neural code for itch while higher-frequency
firing signals pain.
Although we found very few proritogen-selective neurons, this may have
been due to a sampling bias. It was recently reported that a
subpopulation of cat spinothalamic tract neurons in lamina I with
slowly conducting axons responded to cutaneous histamine over a time
course similar to that of histamine-evoked itch in humans
(Andrew and Craig 2001). Several properties of these
neurons (mechanical insensitivity; C-fiber afferent input) are
consistent with those of slowly conducting C fibers in humans that
responded to cutaneous iontophoretic application of histamine over a
time course closely matching that of concomitant itch sensation
(Schmelz et al. 1997). However, some of the
histamine-responsive spinothalamic tract neurons also responded to
mustard oil (Andrew and Craig 2001), and more work is
needed to assess the chemical selectivity of these neurons.
Nevertheless, these findings are consistent with specificity theory.
This receives further support from observations that cutaneous or
intraneural stimulation can elicit itch, the intensity of which
increases with frequency but never becomes painful (Schmidt et
al. 1993; Tuckett 1982). Conversely,
intraneural microstimulation can elicit pain that does not become itch
at low stimulus frequencies (Handwerker et al. 1991;
Ochoa and Torebjork 1989). Overall, the available
evidence appears to favor the idea of a specific itch pathway. However,
the sizable population of dorsal horn neurons that respond
nonselectively to pruritic and algesic stimuli cannot be overlooked. It
is conceivable that both chemically specific and nonspecific pathways
signal itch, analogous to the roles of WDR and NS neurons in signaling
pain (Mayer et al. 1975; Price and Mayer
1975).
Scratching and shaking behavior
Of the irritants studied, only 5-HT elicited significant
scratching and shaking in a dose-dependent manner. Formalin elicited significant scratching and shaking, but it was not dose related and was
less than that evoked by 5-HT. Capsaicin elicited sporadic scratching
that was insignificant at all doses tested. Histamine elicited almost
no scratching or shaking at concentrations up to 10%. Our results are
reasonably consistent with data from Kuraishi's group showing that
5-HT elicited dose-related scratching (Yamaguchi et al.
1999), while neither histamine, capsaicin, nor Formalin elicited scratching in ddY mice (Kuraishi et al. 1995;
Yamaguchi et al. 1999), although histamine elicits
scratching in ICR mice (Kitagawa et al. 1997) and
hairless guinea pigs (Woodward et al. 1995).
Conceivably, histamine may be algesic rather than pruritic in rats,
compared with humans in whom histamine is pruritic but can elicit pain
when injected into deeper skin layers and/or at higher concentrations
(Keele and Armstrong 1964). These data indicate that
5-HT is a more effective pruritogen than histamine in some rodent strains.
Rodent mast cells contain 5-HT (Graziano 1988;
Gustafsson 1980; Purcell et al. 1989),
and unmyelinated nerve endings express 5-HT2 receptors at the dermal-epidermal junction
(Carlton and Coggeshall 1997) where itch is
evoked in humans (Keele and Armstrong 1964;
Shelley and Arthur 1957). 5-HT is less pruritic than
histamine in humans (Fjellner and Hägermark
1979; Hägermark 1995;
Weisshaar et al. 1997), but the reverse may hold in
rodents. Another interpretation is that ic 5-HT evokes pain.
Intraplantar 5-HT produces inflammation and hyperalgesia (Sufka
et al. 1992; Tokunaga et al. 1998). Behavioral responses to the algogen Formalin are potentiated by 5-HT and blocked
by 5-HT antagonists (Abbott et al. 1996,
1997; Giordano and Rogers 1989). In mice,
5-HT elicited scratching via a 5-HT2 receptor
(Yamaguchi et al. 1999). The relationship between
scratching and 5-HT dose was bell-shaped, and it may be speculated that
5-HT is pruritic at lower doses but becomes painful at higher doses where scratching behavior decreases.
5-HT also elicited "wet dog" shakes reminiscent of opiate withdrawal and possibly related to grooming behavior. Shaking behavior correlated well with scratching (Fig. 4, A and D) and therefore may provide another useful parameter to assess chemogenic sensations.
We observed significant scratching with 10 µl ic Formalin. The
Formalin pain test (Dubuisson and Dennis 1977) usually
involves larger subcutaneous (sc) hindpaw injections (50 µl).
Kuraishi et al. (1995) did not observe scratching in
mice following sc Formalin (100 µl) in neck skin, which may have
induced pain. In human studies noxious chemicals in superficial layers
of skin often evoke itch, while they are more likely to be painful in the dermis or subdermis (Keele and Armstrong 1964;
Shelley and Arthur 1957). Furthermore, itch may be
suppressed by ic injection volumes >50 µl (Arthur and Shelley
1959; Keele and Armstrong 1964). The
Formalin-induced scratching observed presently usually began 8-10 min
postinjection, coinciding with the interphase and ending at the
beginning of the second phase of activity elicited by intraplantar Formalin in C fibers (McCall et al. 1996) and dorsal
horn neurons (Chapman and Dickenson 1995;
Dickenson and Sullivan 1987). That scratching occurred
at the nadir of Formalin-induced nociception indirectly supports the
idea that scratching reflects itch rather than pain.
Capsaicin elicits burning pain and hyperalgesia in humans
(LaMotte et al. 1991; Schmelz et al.
2000) by binding to C fiber VR-1 receptors (Caterina et
al. 1997, 2000). Topical capsaicin can also
elicit itch (Green 1990; Green and Shaffer
1993). Presently, capsaicin elicited scratching in some rats,
possibly reflecting itch dependent on the exact ic location of the
injection needle. Future behavioral studies assessing itch in animals
should take into account factors including concentration, volume, skin
site, and method of application, which may all influence the degree of
resulting itch or pain.
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ACKNOWLEDGMENTS |
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The authors gratefully acknowledge M. I. Carstens for histology and for assistance in some of the behavioral studies.
This work was supported by grants from the California Tobacco-Related Disease Research Program (6RT-0231) and the National Institute of Neurological Disorders and Stroke (NS-35788).
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FOOTNOTES |
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Address for reprint requests: E. Carstens, Section of Neurobiology, Physiology and Behavior, University of California, 1 Shields Ave., Davis, CA 95616 (E-mail: eecarstens{at}ucdavis.edu).
Received 25 May 2001; accepted in final form 31 October 2001.
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ABSTRACT
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