Section of Neurobiology, Physiology and Behavior, University of California, Davis, California 95616
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ABSTRACT |
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Jinks, Steven L. and E. Carstens. Superficial Dorsal Horn Neurons Identified by Intracutaneous Histamine: Chemonociceptive Responses and Modulation by Morphine. J. Neurophysiol. 84: 616-627, 2000. We have investigated whether neurons in superficial laminae of the spinal dorsal horn respond to intracutaneous (ic) delivery of histamine and other irritant chemicals, and thus might be involved in signaling sensations of itch or chemogenic pain. Single-unit recordings were made from superficial lumbar dorsal horn neurons in pentobarbital sodium-anesthetized rats. Chemoresponsive units were identified using ic microinjection of histamine (3%, 1 µl) into the hindpaw as a search stimulus. All superficial units so identified [9 nociceptive-specific (NS), 26 wide-dynamic-range (WDR)] responded to subsequent ic histamine. A comparison group of histamine-responsive deep dorsal horn neurons (n = 16) was similarly identified. The mean histamine-evoked discharge decayed to 50% of the maximal rate significantly more slowly for the superficial (92.2 s ± 65.5, mean ± SD) compared with deep dorsal horn neurons (28.2 s ± 11.6). In addition to responding to histamine, most superficial dorsal horn neurons were also excited by ic nicotine (22/25 units), capsaicin (21/22), topical mustard oil (5/6), noxious heat (26/30), and noxious and/or innocuous mechanical stimuli (except for 1 unit that did not have a mechanosensitive receptive field). Application of a brief noxious heat stimulus during the response to ic histamine evoked an additive response in all but two cases, followed by transient depression of firing in 11/20 units. Intrathecal (IT) administration of morphine had mixed effects on superficial dorsal horn neuronal responses to ic histamine and noxious heat. Low morphine concentrations (100 nM to 1 µM) facilitated histamine-evoked responses (to >130% of control) in 9/24 units, depressed the responses (by >70%) in 11/24, and had no effect in 4. Naloxone reversed morphine-induced effects in some but not all cases. A higher morphine concentration (10 µM) had a largely depressant, naloxone-reversible effect on histamine responses. Responses of the same superficial neurons to noxious heat were facilitated (15/25), reduced (8/25), or unaffected (2/25) by low morphine concentrations and were depressed by the higher morphine concentration. In contrast, deep dorsal horn neuronal responses to both histamine and noxious heat were primarily depressed by low concentrations of morphine in a naloxone-reversible manner. These results indicate that superficial dorsal horn neurons respond to both pruritic and algesic chemical stimuli and thus might participate in transmitting sensations of itch and/or chemogenic pain. The facilitation of superficial neuronal responses to histamine by low concentrations of morphine, coupled with inhibition of deep dorsal horn neurons, might underlie the development of pruritis that is often observed after epidural morphine.
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INTRODUCTION |
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The sensation of pain is
thought to be signaled by both nociceptive-specific (NS) and wide
dynamic range (WDR)-type neurons in the spinal dorsal horn
(Willis 1985). Available evidence indicates that WDR
neurons, which are common in the deep laminae of the dorsal horn, are
sufficient for pain perception (Mayer et al. 1975
;
Price and Mayer 1975
) and encode the intensity of
noxious stimuli in a behaviorally relevant manner (Maixner et
al. 1986
). NS and WDR neurons in the superficial dorsal horn
undoubtedly also play an important role in pain mechanisms. Neurons in
lamina I of the superficial dorsal horn contribute appreciably to
ascending sensory pathways, such as the spinothalamic tract that
signals pain, temperature, and itch sensations (White and Sweet
1969
; Willis 1985
). The superficial
dorsal horn is the primary termination area for nociceptive primary
afferent fibers, and neurons in laminae I and the substantia gelatinosa
respond to acute noxious stimuli (Bennett et al. 1980
;
Cervero et al. 1979
; Christensen and Perl 1970
; Light et al. 1979
). A role for superficial
dorsal horn neurons in chronic pain was demonstrated recently using
targeted neurotoxic ablation of neurokinin-1 receptor-expressing
neurons (Nichols et al. 1999
). This markedly reduced
behavioral manifestations of chronic pain, although behavioral
responses to acute noxious stimuli appeared to be normal
(Nichols et al. 1999
), thus demonstrating the importance
of these superficial neurons in certain aspects of pain. Our laboratory
has recently begun to investigate the role of dorsal horn neurons in
chemogenic mechanisms of itch, irritation, and pain. WDR neurons in the
deep dorsal horn respond to intracutaneous (ic) microinjection of both
pruritic and algesic chemicals (Carstens 1997
;
Jinks and Carstens 1998a
,b
, 1999
;
Li et al. 1995
; Wei and Tuckett 1991
).
Because of the potential importance of the superficial dorsal horn in
pain, we have presently investigated whether neurons in this region
respond to pruritic and algesic chemical stimuli in a manner that is
relevant to chemogenic sensations of itch or pain.
Itch (pruritis) is a debilitating symptom of many pathological skin
conditions. However, little is known about the neural pathways that
convey itch, and how these pathways may differ from, or integrate with,
pain pathways. Available psychophysical evidence indicates that pain
and itch are signaled by separate neural populations. Itch is said to
rarely coexist with pain, and noxious stimuli inhibit itch
(Graham et al. 1951; Greaves and Wall
1996
; McMahon and Koltzenburg 1992
; Ward
et al. 1996
). Furthermore, administration of opiates by
systemic, epidural, or intrathecal (IT) routes reduces pain but often
elicits itching (Brennum et al. 1993
; Bromage et al. 1982a
; Fischer et al. 1988
; Hales
1980
; for reviews see Ballantyne et al. 1988
;
Bromage 1981
; Morgan 1987
).
Microneurographic studies have revealed that intraneural electrical
microstimulation near the axon(s) of polymodal nociceptors elicits a
sensation of pain that increases in intensity with increasing stimulus
frequency, but does not change to itch at lower frequencies
(Ochoa and Torebjork 1989
). Conversely, intraneural
microstimulation occasionally elicits a sensation of itch that
increases in intensity but does not become painful at higher
stimulation frequencies (Schmidt et al. 1993
), nor does
increasing the frequency of electrical stimulation at a cutaneous
"itch" spot evoke pain (Tuckett 1982
). These data suggest that itch is conveyed by its own specific sensory pathway ("specificity" theory). In support of this, a population of slowly conducting sensory fibers innervating human skin was recently found to
respond to cutaneous application of histamine, an itch-producing chemical (Broadbent 1955
; Hagermark 1995
;
Handwerker et al. 1987
, 1991
;
Keele and Armstrong 1964
; Magerl and Handwerker
1988
; Rothman 1941
; Shelly and Arthur
1957
; Simone et al. 1987
, 1991
;
Ward et al. 1996
; Yosipovitch et al.
1996
), over a time course that matched the concomitant
sensation of itch (Schmelz et al. 1997
). It is unknown
whether these putative "itch" fibers connect to a central pathway
that faithfully conveys their responses. Our previous studies have
shown that ic histamine evokes responses in deep dorsal horn WDR
neurons, but that these neurons also respond to noxious stimuli that
would normally be painful (Carstens 1997
). One aim of
the present study was to determine whether neurons exist in the
superficial dorsal horn that selectively respond to ic histamine. To
accomplish this, we developed a strategy of searching for active units
in the superficial dorsal horn following ic injection of histamine. The
rationale was to identify potential histamine-responsive neurons
without delivering any other stimuli to the skin that might inhibit
itch neurons. Furthermore, this strategy would also identify neurons
that only respond to chemical stimuli and do not possess a mechanical
receptive field, as is the case for some histamine-responsive afferent
fibers (Schmelz et al. 1997
). Once identified in this
manner, the dorsal horn units were tested for responses to additional
ic histamine, as well as to other algesic chemicals to assess the
degree of chemical selectivity.
Although contemporary evidence favors the specificity theory for itch,
other theories are still viable. The intensity theory postulates that
itch and pain are conveyed by a common population of WDR neurons that
signal itch at a low firing frequency and pain at higher frequencies
(Magerl 1996; McMahon and Koltzenburg 1992
). Our observation that deep dorsal horn WDR units respond to both pruritic (histamine) and noxious (heat, capsaicin) stimuli is
consistent with this. Until the existence of an itch-specific pathway
is proven, intensity and related theories of itch (see Carstens
1997
; Handwerker 1992
; LaMotte
1992
) cannot be discounted.
The final aim of this study was to investigate the effects of morphine
on histamine-responsive dorsal horn neurons. As noted earlier, epidural
and IT administration of morphine at analgesic doses frequently causes
pruritis, and intracranial administration of opiates elicits scratching
behavior in animals (Koenigstein 1948; Thomas and
Hammond 1995
; Thomas et al. 1992
; Tohda
et al. 1997
; Yamaguchi et al. 1998
), suggestive
of itch. It may therefore be hypothesized that opiates excite or
facilitate itch-signaling spinal neurons. We reported previously that
systemic morphine significantly attenuated responses of deep dorsal
horn WDR units to ic histamine (Carstens 1997
). Other
studies have shown that low doses of morphine facilitate C
fiber-evoked responses of some superficial dorsal horn neurons, while
inhibiting responses of neurons in deeper laminae (Dickenson and
Sullivan 1986
; Magnuson and Dickenson 1991
;
Sastry and Goh 1983
; Woolf and Fitzgerald 1981
). In the present study we wished to investigate effects of IT morphine on responses of superficial and deep dorsal horn NS and WDR
units to ic histamine and noxious heat. We hypothesize that low doses
of morphine should facilitate responses of superficial neurons and
depress deep neurons, while higher doses would have a uniform
depressant effect. An abstract of this work has appeared (Jinks
and Carstens 1999
).
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METHODS |
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This study was approved by the University of California Davis
Animal Use and Care Advisory Committee. The methods for single-unit recording and chemical stimulation of the skin are the same as in our
previous studies (Carstens 1997; Jinks and
Carstens 1998a
,b
, 1999
) and are summarized here,
with novel methods described in more detail. Thirty-seven adult male
Sprague-Dawley rats were anesthetized with pentobarbital sodium
(induction: 65 mg/kg ip; maintenance: 10-20 mg · kg
1 · h
1 iv via a
jugular cannula), and a laminectomy was performed to expose the lumbar
spinal cord for single-unit recording. The spine was secured in a
stereotaxic frame, and the spinal cord was covered with agar. After the
agar hardened, a small opening was created to form a pool filled with
0.9% saline over the lumbar enlargement. A tungsten microelectrode was
used to obtain extracellular single-unit recordings. We used electrodes
with higher impedances (13-18 M
; F. Haer) than in our previous
studies to better isolate potentially small units. Unit responses to ic
chemical or noxious heat stimuli were collected using custom software
(Forster and Handwerker 1990
) and quantified as the
total number of impulses/60- or 150-s period, or by maximal firing
frequency. A 31-gauge injection needle connected to a microsyringe was
inserted into the dermis in either the heel or lateral toe of the hind
paw (Carstens 1997
).
To search for units, histamine (3% = 163 mM) was microinjected (1 µl) at the ic site, and the microelectrode was introduced in 10-µm
steps into the ipsilateral superficial lumbar dorsal horn using a
hydraulic microdrive to search for spontaneously active units. In most
experiments, the search was restricted to a depth of <250 µm below
the cord surface to isolate superficial units. In later experiments we
used the same search strategy to isolate deep dorsal horn units (>250
µm below the surface). When a spontaneously firing single unit was
isolated, we waited for the activity to wane (minimum 10 min). In
approximately 50% of searches, a spontaneously firing unit was
isolated in the first electrode track within 3 min following the ic
histamine search stimulus. If a unit was not isolated in the first
penetration, one or two additional electrode penetrations were made in
the same spinal area, and a unit was usually identified. However, in
approximately 20-25% of experiments, the initial search failed. We
then either 1) reinjected histamine at the same skin site
(>10 min later) and resumed the search in additional electrode
penetrations, or 2) placed the ic histamine injection needle
into a region of the hind paw distant from the previous site (heel vs.
toe), or into the contralateral hind paw. Histamine was then injected, and we searched with electrode penetrations in the topographically corresponding new spinal area. Thus, in the former instance, units were
isolated following a second ic histamine search stimulus at the
identical skin site, although the number of units isolated in this way
was low (<20%). We do not believe that the second histamine search
stimulus had any greater effect than the first one on the unit's
subsequent responses to histamine, for the following reasons. First,
unit responses to multiple ic histamine injections repeated at 10-min
interstimulus intervals did not exhibit pronounced tachyphylaxis or
sensitization (Fig. 3A). Second, we previously showed that
deep dorsal horn WDR unit responses isolated using a mechanical search
strategy did not show significant tachyphylaxis or sensitization to
repeated ic histamine injections (Carstens 1997;
Carstens and Jinks 1998a
).
A potential drawback of this technique is that it is not certain whether a unit's ongoing activity was elicited by ic histamine or was truly spontaneous. That all units isolated in this manner 1) showed a time-dependent decrease in spontaneous firing, and 2) responded to a subsequent ic histamine stimulus, indicates that the firing was histamine dependent. We normally rejected units whose spontaneous firing did not decline. However, a small number of units exhibiting constant spontaneous activity were tested with a second ic histamine injection, and none responded. Therefore we believe that the present search strategy was efficient in isolating histamine-responsive units.
We then tested whether the unit responded to ic histamine by making a second microinjection at the same ic site. An example of a spontaneously firing unit isolated in this manner, and its response to the subsequent histamine injection, is shown in Fig. 1. Following histamine, unit activity was recorded for variable periods until the firing declined to the prehistamine level. At this point, one of the following was done (not necessarily in the order shown).
1) Duration of response to histamine and tacyhphylaxis. The unit's responses to additional ic histamine injections at a 10-min interstimulus interval were recorded to test for tachyphylaxis or sensitization. For histamine and other chemicals (see 5 below), a unit was considered to respond if its firing rate increased by more than 200% above the spontaneous level prior to chemical stimulation. Responses were quantified by counting the total number of impulses/150 s following each histamine stimulus, averaged for all units across trials, and compared using ANOVA, with P < 0.05 considered significant. For the averaged peristimulus time histograms (PSTHs) shown in Fig. 4, units' responses in each 1-s time bin were averaged.
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The duration of each superficial and deep dorsal horn unit's initial response to ic histamine was determined as follows. A second-order polynomial function was fitted to the decay in the unit's ic histamine-evoked firing rate using commercial software (Microcal Origin). The time for the response to decay to 50% of the peak firing rate was taken from this curve, averaged for all superficial and deep units, and compared using an unpaired t-test with P < 0.05 taken as significant.
2) Receptive field mapping and unit classification. The unit's mechanosensitive receptive field was mapped crudely by hand, and then more precisely with von Frey filaments. This was only done after the unit's response to histamine had been recorded in the absence of any prior physical stimulation of the hindpaw. WDR units were classified by their response to light tactile stimulation (von Frey filament with 0.7-g bending force) and additionally to noxious heat and firm but nondamaging pinch with forceps. The few units that did not respond to noxious heat, but responded to innocuous and noxious mechanical stimuli as well as ic histamine, were therefore classified as WDR. NS units were classified by lack of response to light tactile stimulation (von Frey filament with 0.7-g bending force), but responded to noxious heat and firm but nondamaging pinch with forceps. One unit did not respond to pinch but did to noxious heat and histamine and was included as NS.
3) Response to noxious heat. The unit's responsiveness to a noxious heat stimulus (52°C, 5-s duration from adapting temperature of 35°C, using a Peltier thermode having a contact area of 1 cm2) delivered to the low-threshold region of the mechanosensitive receptive field (or at the ic injection site) was tested. In some cases, successive stimuli were delivered at >5-min interstimulus intervals to determine whether sensitization occurred. Responses were quantified by counting the total number of impulses/60 s following heat onset, averaged for all units, and successive trials compared using ANOVA.
4) Effect of heat on histamine response. Histamine was injected ic, followed 60 s later by application of the noxious heat stimulus to determine whether heat influenced the histamine-evoked discharge. The thermode was positioned against the skin over the site of the ic microinjection needle.
5) Response to other irritants. Additional chemicals were injected ic via separately placed 31-gauge needles (except mustard oil, which was delivered topically to the skin surface). Chemicals tested were as follows: capsaicin (330 µM, diluted from a 1% stock solution in 70% ethanol; Sigma), nicotine (60 or 600 mM in saline; Sigma), or mustard oil (10% = 1 M; Fluka).
6) Morphine. The effect of IT morphine sulfate (100 nM to 10 µM in 0.9% saline) was tested on unit responses to ic histamine noxious heat, recorded alternately at >5-min interstimulus intervals. The heating thermode was positioned over the ic microinjection needle. The saline was removed from the pool over the spinal cord, and morphine was delivered IT by syringe in a volume of approximately 0.5 ml to the cord surface. Responses recorded at various times following a given concentration of IT morphine were averaged and compared with the same units' mean response prior to morphine using repeated-measures ANOVA with P < 0.05 considered to be significant. The morphine solution was removed to allow IT administration of naloxone (10 µM) in the same manner. Mean responses at various times following naloxone were compared with mean pre- and postmorphine responses (paired t-test). Only one unit/rat was tested with morphine.
In most experiments only one unit was recorded. In four experiments, two units were recorded on opposite sides of the spinal cord, and in five experiments two units were recorded on the same side of the spinal cord but having disparate receptive fields (e.g., heel vs. distal toe).
At the conclusion of the experiment, an electrolytic lesion was made at the spinal recording site by passing 6-V DC through the electrode for 20 s, and the rat was killed by overdose with pentobarbital. The spinal cord was removed and fixed in 10% buffered Formalin. At least 7 days later, the spinal cord was cut in 50-µm frozen sections, counter-stained with neutral red, and examined under the light microscope to identify lesioned recording sites.
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RESULTS |
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Unit sample
A total of 51 units (10 NS, 41 WDR) were isolated using the ic histamine search strategy. Thirty-five units (26 WDR, 9 NS) were classified as "superficial" (mean depth: 155.5 ± 120.5 µm, mean ± SD), and 16 units (15 WDR; 1 NS) were classified as "deep" (mean depth: 534.7 ± 63.5 µm). Recording sites are shown in Fig. 4 for superficial (Fig. 4A) and deep (Fig. 4B) dorsal horn neurons.
All units were initially identified by their spontaneous firing following ic histamine injection into the plantar surface of the ipsilateral hind paw (Fig. 1). Spontaneous activity measured >10 min after the initial histamine search stimulus was 0-3 Hz in 66% of all units, 3-8 Hz in 28%, and 8-13 Hz in 6%. Mean spontaneous activity in superficial neurons (2.43 ± 2.47 Hz) was not significantly different (P > 0.05; unpaired t-test) from mean spontaneous activity in deep neurons (3.8 ± 3.87 Hz).
After isolating units and testing for responses to additional histamine stimuli (see next section), mechanosensitive receptive fields and responses to noxious heat were determined. All WDR units had low-threshold mechanosensitive receptive fields ranging from small (1-3 toes, 59%), to medium (encompassing the heel, 27%), to large (>half of the ventral hind paw surface, 14%). Figures 1 and 5 show examples of low-threshold mechanosensitive receptive fields. Receptive fields of superficial WDR neurons tended to be smaller than those of deep WDR neurons, although this was not systematically quantified. Most of the NS units had high-threshold mechanical receptive fields, although one did not have an identifiable mechanosensitive receptive field. This unit, shown in Fig. 2, responded to ic histamine as well as capsaicin and nicotine and responded weakly to noxious heat.
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Responses to repeated ic histamine and noxious heat: lack of tachyphylaxis
After a unit was isolated, we waited >10 min until its
spontaneous firing declined to a low, steady level, at which point we
recorded its response to a second ic histamine injection (see Fig. 1).
All units responded to the second ic histamine injection, most showing
a rapid increase in firing rate within 1-2 s, followed by a gradual
decline (Figs. 1, 2, and 5). That we did not find histamine-unresponsive units indicates that the initial histamine stimulus did not completely desensitize the skin. We previously reported that deep WDR neuronal responses to repeated ic histamine do
not exhibit tachyphylaxis (Carstens 1997) and verified
this presently for superficial units. Figure
3A plots mean responses of 15 superficial units to ic histamine repeated at a 10-min interstimulus interval. There was no significant decline or increase (sensitization) in successive responses (2-factor ANOVA, P > 0.05).
Since heat alternated with ic histamine stimuli, this result indicates
that histamine had no significant sensitizing or desensitizing effect on heat-evoked responses. However, using the present strategy we were
unable to directly compare mechanically or thermally evoked responses
of the same unit before and after the initial histamine search
stimulus. We previously showed that an initial ic injection histamine
caused a small but significant expansion at the fringe of the
low-threshold portion of cutaneous receptive fields of deep WDR neurons
(Carstens 1997
; Jinks and Carstens
1998a
).
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Noxious heat (52°C, 5 s) evoked responses in 38/42 units tested (27/30 superficial and 11/12 deep). Of the four units not excited by heat, two superficial units were unresponsive, and in two units (1 superficial and 1 deep) the spontaneous activity was inhibited by the heat stimulus. There was a slight increasing trend in successive mean responses of superficial units to noxious heat applied at a 10-min interstimulus interval (Fig. 3B), but this was not statistically significant (2-factor ANOVA, P > 0.05).
Time course of responses to histamine and noxious heat
The left column of Fig. 4 shows averaged PSTHs of histamine-evoked responses of superficial (Fig. 4A) and deep (Fig. 4B) neurons. The maximal firing rate of superficial units was significantly lower compared with deep neurons, and the time to decay to 50% of the maximal response was significantly longer for superficial units. Parameters of histamine-evoked responses of superficial and deep dorsal horn units are provided in Table 1.
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The right column of Fig. 4 shows averaged PSTHs of the same superficial (Fig. 4A) and deep (Fig. 4B) units to noxious heat. Although the peak response of the deep units was larger compared with the superficial units, there were no statistically significant differences in parameters describing the magnitude or time course of heat-evoked responses for these two populations (Table 1).
Effect of noxious heat on histamine-evoked discharge
In 20 superficial neurons, a noxious heat stimulus (52°C, 5 s) was delivered to the hind paw at the histamine injection site 60 s following ic histamine. The heat stimulus evoked a response that summed with the histamine discharge in all but two cases. This was followed by transient depression of firing in 11/20 units that lasted for 10-60 s. An example is shown in Fig. 5B.
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Response to other irritant chemicals
Histamine-responsive superficial units were tested for responsiveness to nicotine, capsaicin, and mustard oil. All units responded to one or more of the additional chemicals tested (Table 2). Figure 2 shows an example of a NS unit with no cutaneous mechanical receptive field that responded to ic histamine, capsaicin, and nicotine. Figure 5 shows a typical example of a superficial WDR unit that responded to histamine (Fig. 5A), noxious heat (Fig. 5B), and capsaicin, mustard oil, and nicotine (Fig. 5C). Overall, 94% of superficial units responded to nicotine, 95% to capsaicin and 83% to mustard oil (Table 2).
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Morphine effects on unit responses to histamine and heat
SUPERFICIAL UNITS. Low concentrations of IT morphine (100 nM or 1 µM) had mixed effects on superficial unit responses to histamine and heat, and morphine did not always affect an individual unit's responses to histamine and heat in the same manner. Low-dose morphine facilitated histamine-evoked responses (by >130% of the baseline response) in 9/24 units, depressed histamine-evoked responses (below 70% of the baseline response) in 11/24 units, and had no effect in 4. A typical example is shown in Fig. 6. The response of this superficial WDR unit to ic histamine was facilitated by low concentrations of IT morphine (Fig. 6A, 2nd and 3rd PSTHs from left) and was suppressed by the higher (10 µM) concentration in a naloxone-reversible manner (Fig. 6A, right 2 PSTHs). Similar effects of morphine and naloxone were observed for this unit's responses to noxious heat (Fig. 6B).
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DEEP DORSAL HORN UNITS. In contrast to superficial dorsal horn units, application of low-concentration morphine (100 nM to 1 µM) almost uniformly suppressed responses of deep units to both histamine and heat in a naloxone-reversible manner (Fig. 7, E and F). Following 100 nM and 1 µM morphine, histamine-evoked responses were significantly lower compared with premorphine levels (2-factor ANOVA, P < 0.011). Twenty minutes following naloxone, histamine-evoked responses were no longer significantly different from premorphine responses (paired t-test, P > 0.05) but were significantly greater than the previous response following low-concentration morphine (P < 0.042). A similar analysis revealed that deep unit responses to noxious heat were significantly reduced following 100 nM and 1 µM morphine (2-factor ANOVA, P < 0.0026), and that following naloxone the mean response recovered such that it was significantly different from postmorphine (paired t-test, P < 0.036) but not from the premorphine control level (paired t-test, P > 0.05). There were no significant changes in spontaneous activity after morphine application, nor were changes in spontaneous activity correlated with changes in the units' response to histamine or heat.
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DISCUSSION |
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The present results confirm and extend our earlier studies
(Carstens 1997; Jinks and Carstens
1998a
,b
, 1999
) by showing that ic
histamine excites NS and WDR neurons in the superficial as well as deep
dorsal horn that were isolated using a novel ic histamine search
strategy. All superficial histamine-responsive units also responded to
one or more additional algesic chemical stimuli and thus cannot be
considered histamine specific. The duration of histamine-evoked
responses of superficial units was variable and significantly longer
compared with deep dorsal horn units, suggesting a role for these
neurons in signaling chemogenic pain or itch sensations. Finally, IT
morphine was shown to have variable effects on histamine- and noxious
heat-evoked responses of superficial dorsal horn units. Low
concentrations of morphine usually had either a facilitatory or
depressant effect, which was naloxone reversable in some (but not all)
cases, while a higher concentration had a more uniform depressant
effect. This contrasts with a uniformly depressant effect of low
morphine concentrations on deep dorsal horn units. We wish to discuss
these results in terms of methodology, the role of chemonociceptive
neurons in itch and pain sensations, and their modulation by opioids.
Methodological considerations
The present study employed a novel search strategy to isolate
neurons exhibiting spontaneous firing following delivery of an ic
histamine search stimulus to the skin. This strategy proved to be
efficient in isolating superficial dorsal horn units that gave
prolonged excitatory responses to subsequent ic histamine. An advantage
of this strategy is that we could test effects of histamine in skin
that had not received prior stimulation. A disadvantage, however, is
that we cannot assess the possible effect of the initial histamine
stimulus on unit response properties that were only determined after
repeated ic histamine stimuli had been delivered. We do not believe
that the initial histamine search stimulus had a major effect on
subsequent responses to histamine, because repetitive delivery of
histamine did not result in tachyphylaxis or sensitization of responses
in the present study (Fig. 3A). Furthermore, in our prior
study of deep dorsal horn WDR units isolated by a mechanical search
strategy, we did not observe tachyphylaxis to repeated ic histamine
injections (Carstens 1997). It is quite possible, however, that the histamine search stimulus may have affected mechanical receptive fields or thermal sensitivity of the superficial units, since we previously reported that an initial ic histamine stimulus produced a small but significant expansion at the fringe of
the low-threshold region of mechanosensitive receptive fields in deep
WDR units (Carstens 1997
; Jinks and Carstens
1998a
). This might result in an overestimation of the
low-threshold mechanical sensitivity and/or incidence of WDR units in
the superficial dorsal horn. Nonetheless, using the present strategy we
found one unit that had no mechanosensitive receptive field (Fig. 2).
Thus we believe that this search strategy may prove fruitful in
identifying chemonociceptive dorsal horn neurons.
We presently did not test whether the dorsal horn units projected in
ascending pathways such as the spinothalamic tract. A recent study
reported that a small fraction of identified spinothalamic tract
neurons in lamina I of the superficial dorsal horn in cats gave
prolonged responses to histamine applied iontophoretically to the hind
paw skin (Andrews and Craig 1999). The ascending axons of these units were very slowly conducting, suggesting that the neurons
might be quite small. Our ic histamine search strategy would be biased
toward finding larger cell bodies, but perhaps incorporating antidromic
stimulation with the ic chemical search stimulus would improve the
chances of finding potentially small chemonociceptive neurons.
Chemonociceptive responses in relation to itch and pain
Previous studies have shown that C-fiber polymodal nociceptors can
respond to histamine as well as algesic chemicals such as capsaicin
(Baumann et al. 1991; Davis et al. 1993
;
Handwerker et al. 1991
; Kress et al.
1992
; LaMotte et al. 1988
; Schmelz et al.
1997
). A small fraction of these chemonociceptive C-fibers did
not respond to noxious mechanical and/or thermal stimuli, including a
recently identified population of very slowly conducting C-fibers in
human skin that gave prolonged discharges to histamine (Schmelz
et al. 1997
, 1998
). One rationale for presently
using the ic histamine strategy was to determine whether some spinal units only respond to histamine and not thermal or mechanical stimuli,
thus faithfully conveying input from the histamine-responsive C-fibers
in a specific "itch" pathway. However, nearly all of the present
units could be classified as NS or WDR; only one potential "chemospecific" unit did not have a mechanical receptive field. Using antidromic stimulation, Andrews and Craig (1999)
have identified a small proportion of spinothalamic tract units in
lamina I that lacked mechanical receptive fields but responded to
histamine. Thus there is evidence for "pure" chemonociceptive
spinal neurons, but it is not yet clear how large this population is
compared with the apparently sizable population of NS and WDR neurons
that respond to histamine.
The present histamine-evoked discharges of superficial units lasted
significantly longer, sometimes for >10 min, compared with deep WDR
units whose responses rarely lasted >2-3 min. The longer time course
of superficial unit responses to histamine is comparable to prolonged
histamine-evoked responses of slowly conducting C-fibers in human skin
(Schmelz et al. 1997) and was in the lower range (8-12
min) of histamine-elicited itch sensation in humans (Bickford
1938
; Fruhstorfer et al. 1986
;
Heyer et al. 1997
; Simone et al. 1987
,
1991
; Ward et al. 1996
;
Yosipovitch et al. 1996
). Therefore some of the
presently recorded superficial units might be involved in signaling
itch sensation. However, all units additionally responded to other
algesic chemicals (Table 2), and nearly all responded to noxious heat.
In this regard, the histamine-responsive peripheral C-fibers also
responded to capsaicin (Schmelz et al. 1997
). Similarly,
histamine-responsive lamina I spinothalamic units (Andrews and
Craig 1999
) might participate in an "itch"-specific
pathway, but when tested, these units also responded to algesic
chemicals such as mustard oil, and some also to noxious heat. Therefore
the data to date indicate that most, if not all, superficial dorsal
horn units that respond to histamine also respond to algesic chemical
or thermal stimuli. Future work focused on identifying chemonociceptive
units may uncover histamine-specific units that could exclusively
signal itch. Alternatively, there may be sub-classes of
chemonociceptive units with gradations in their responses to pruritic
versus algesic chemical stimuli. Itch may be selectively signaled by
units that respond more vigorously to pruritogens than to algesic
stimuli. In this regard, it is not certain to what extent some of the
presently tested "algesic" chemicals may also have pruritogenic
properties. While mustard oil on the skin elicits a sensation of
burning pain, topical capsaicin has been reported to elicit itch in
humans (Green 1990
; Green and Shaffer
1993
), and cholinergic agonists excite some histamine-sensitive C-fibers (Schmelz et al. 1998
) and induce variable
degrees of itch and burning pain sensation (Magerl et al.
1990
). More work is needed to characterize the pruritic versus
algesic properties of capsaicin, nicotine, and other skin irritants in
relation to their effects on pain and itch sensory systems.
The present results do not rule out the possibility of additional itch
mechanisms, such as frequency or occlusion (see
INTRODUCTION). While the psychophysical evidence suggests
that itch and pain are signaled separately, our electrophysiological
data indicate that nearly all dorsal horn units respond to both
pruritogenic and algesic stimuli. A possible clue is the more prolonged
discharge of superficial versus deep dorsal horn units to histamine.
One might speculate that the deep units signal pain (Price and
Mayer 1975), while a proportion of superficial units signal
itch, at least under certain conditions. Pain would be signaled by
activity in the deep units, and any concomitant sensation of itch would be suppressed by occlusion (Handwerker 1992
) or by
inhibition of superficial units (although we know of no evidence for
inhibition of superficial units by deep dorsal horn units). Pruritogens
activate both sets of units. However, the response of deep units to
pruritogens is brief, so that any suppression of itch by activity in
deep units is short-lasting, permitting a more prolonged itch signal to
be conveyed by superficial units. This is consistent with reports that
ic histamine sometimes evokes brief pain, followed by itch (Keele and Armstrong 1964
). The speculative mechanism
suggested here, and frequency coding, are not necessarily mutually
exclusive. Superficial units, which respond to both pruritic and
algesic stimuli, could conceivably signal itch at a low firing
frequency and pain at higher frequencies, in parallel with a separate
dedicated pain pathway.
The preceding arguments assume that histamine is pruritic in rodents,
as it is in humans. However, this may not necessarily be the case based
on studies of scratching behavior (Kuraishi et al. 1995;
but see Woodward et al. 1995
). It would therefore be
worthwhile to test whether other candidate pruritogens, such as
substance P, which induces reliable scratching in rodents
(Kuraishi et al. 1995
), induce prolonged discharges in
superficial dorsal horn units possibly commensurate with a role in
signaling itch.
Cutaneous application of noxious heat usually evoked an excitatory
response that summed with the ongoing histamine-evoked discharge, often
followed by a brief suppression in firing (Fig. 5B) as
previously reported for deep WDR units (Carstens 1997). While this postexcitatory depression might relate to mechanisms of itch
suppression by pain discussed earlier, the time course appears to be
too brief to account for the much more prolonged reduction of itch
sensation by noxious heat and other counterstimuli reported in human
psychophysical studies (Bickford 1938
;
Fruhstorfer et al. 1986
; Gammon and Starr
1941
; Murray and Weaver 1975
; Ward et al.
1996
). This does not necessarily mitigate against a role for
superficial units in itch, however, since prolonged suppression of itch
sensation by counterirritation might involve neural mechanisms above
the level of the spinal dorsal horn.
Effects of morphine
Low concentrations of IT morphine (100 nM and 1 µM) had either
facilitatory or depressant effects on superficial unit responses to ic
histamine and to noxious heat, while a higher morphine concentration (10 µM) consistently depressed responses. These data are consistent with previous reports that µ-opiate agonists exerted excitatory (Craig and Hunsley 1991; Dickenson and Sullivan
1986
; Jones et al. 1990
; Magnuson and
Dickenson 1991
; Sastry and Goh 1983
;
Willcockson et al. 1986
; Woolf and Fitzgerald
1981
) and inhibitory (Craig and Serrano 1994
;
Glaum et al. 1994
; Kohno et al. 1999
;
Light and Willcockson 1999
; Schneider et al.
1998
) effects on superficial dorsal horn neurons. The present
results are also consistent with earlier reports that a high dose of
systemic morphine (3.5 mg/kg ip) significantly depressed ic histamine-
and noxious heat-evoked responses of deep dorsal horn WDR units
(Carstens 1997
) and histamine-evoked c-fos expression in
the dorsal horn (Yau et al. 1992
). Our
observation that superficial unit responses to histamine and noxious
heat were depressed by a higher morphine concentration after being facilitated by a lower morphine concentration is consistent with earlier studies showing biphasic dose-related effects of IT morphine on
superficial dorsal horn unit responses to electrical C-fiber stimulation (Dickenson and Sullivan 1986
) and of
systemic morphine on nociceptive responses of lamina I neurons
(Craig and Seranno 1994
).
The effects of low-concentration morphine on superficial units were not
consistently reversed by naloxone (Fig. 7). Previous studies have also
reported variability in the effectiveness of naloxone to reverse
morphine actions on superficial dorsal horn units (Willcockson
et al. 1986; Woolf and Fitzgerald 1981
).
However, naloxone itself often had depressant and sometimes
facilitatory effects on superficial dorsal horn unit responses
(Fitzgerald and Woolf 1980
; Jones et al.
1990
; Magnuson and Dickenson 1991
), which may
present a confounding factor in interpreting the present effects of naloxone.
In contrast to superficial neurons, low-concentration morphine
consistently reduced responses of deep dorsal horn units to ic
histamine or noxious heat in a naloxone-reversible manner (Fig. 7,
E and F). These results are consistent with an
earlier study showing that electrical C-fiber-evoked responses of deep
dorsal horn units were unaffected or inhibited by lower doses of IT
morphine, whereas responses of more superficial neurons were
facilitated (Dickenson and Sullivan 1986).
Although we did not presently address the mechanisms of morphine
actions, previous studies provide evidence for both postsynaptic (Glaum et al. 1994; Jeftinija 1988
;
Kohno et al. 1999
; Schneider et al. 1998
;
Yoshimura and North 1983
) and presynaptic inhibitory mechanisms (Jessell and Iversen 1977
; Suarez-Roca
et al. 1992
) for the depressant effects of morphine.
Facilitatory effects of morphine may be mediated by disinhibitory
mechanisms (Fields et al. 1983
; Johnson
and North 1992
; Magnuson and Dickenson 1991
; Pan et al. 1990
), or by presynaptic facilitation
(Suarez-Roca et al. 1992
). Because opioid binding sites
are concentrated in the superficial dorsal horn (Lamotte et al.
1976
), and administration of morphine into the substantia
gelatinosa has a depressant effect on deeper dorsal horn neurons
(Duggan et al. 1977
; Sastry and Goh
1983
), it seems likely that the effects of morphine on both superficial and deep dorsal horn units were manifested by an action at
opioid receptors in the superficial dorsal horn.
Pruritis is a common side-effect of epidural or IT opiates (see
INTRODUCTION) and is more common with morphine compared
with more lipophilic opioids such as fentanyl (Ballantyne et al.
1988; Fischer et al. 1988
). In the present
study, low concentrations of IT morphine resulted in facilitation of
the responses of a substantial fraction of superficial dorsal horn
units to ic histamine or noxious heat, although their spontaneous
activity did not increase significantly. In normal subjects receiving
epidural morphine, the onset of pruritis occurred during the most rapid
rostral spread of analgesia (Bromage et al. 1982a
,b
). It
is conceivable that as morphine diffuses over the spinal cord, it
exerts a facilitatory effect on some superficial dorsal horn neurons
exposed to low concentrations so that their enhanced firing could give
rise to a sensation of itch. Furthermore, if such superficial units
were inhibited by activity in deep dorsal horn neurons,
morphine-induced depression of the latter neurons would disinhibit the
superficial neurons that convey itch, and thus further enhance their signal.
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ACKNOWLEDGMENTS |
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The authors thank M. I. Carstens for expert histological assistance.
This work was supported by National Institute of Neurological Disorders and Stroke Grant NS-35778 and California Tobacco-Related Disease Research Program Grant 6RT-0231.
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FOOTNOTES |
---|
Address for reprint requests: E. Carstens, Section of Neurobiology, Physiology, and Behavior, University of California, Davis, CA 95616 (E-mail: eecarstens{at}ucdavis.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 28 January 2000; accepted in final form 20 April 2000.
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REFERENCES |
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