Superficial Dorsal Horn Neurons Identified by Intracutaneous Histamine: Chemonociceptive Responses and Modulation by Morphine

Steven L. Jinks and E. Carstens

Section of Neurobiology, Physiology and Behavior, University of California, Davis, California 95616


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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).


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 MOmega ; 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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Fig. 1. Isolation of superficial unit using intracutaneous (ic) histamine search stimulus. Shortly after ic histamine, a spontaneously active single unit was isolated. Its spontaneous firing is shown at the indicated times following the initial ic histamine injection. The insets at top show an example of the spike waveform (left), the superficial recording site (middle), and site of ic histamine injection (arrow in right-hand figurine of hind paw). When the spontaneous firing had waned at 25 min after the initial ic histamine, histamine was injected again and elicited a response.

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.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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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Fig. 2. Mechanically insensitive superficial dorsal horn isolated by histamine search stimulus. Peristimulus time histograms (PSTHs) show, from left to right, responses to ic histamine, noxious heat, ic capsaicin, and ic nicotine, respectively. Top left inset shows site of stimuli on paw (arrow). Top right inset shows recording site, and bottom right inset shows example of spike waveform.

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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Fig. 3. Lack of tachyphylaxis or sensitization to repeated ic histamine and noxious heat. A: bar graph shows mean responses of 15 dorsal horn units to 3% ic histamine repeated at 10-min interstimulus interval. Error bars: SE. B: responses of same units in A to noxious heat stimuli (52°C for 5 s) repeated at 10-min interstimulus intervals. Although there was an increasing trend, the 3rd response was not significantly different from the 1st.

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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Fig. 4. Duration of ic histamine-evoked responses: longer in superficial vs. deep dorsal horn. A: superficial units. Left side shows averaged PSTH (+SE) of responses of 30 superficial dorsal horn units to 3% ic histamine (at 1st arrow). Second arrow shows mean time to decay to 50% of peak response. Inset shows histologically recovered recording sites of superficial units, plotted on cross-section of L4 spinal cord taken from the atlas of Paxinos and Watson (1998). Right side shows averaged PSTH of same units to noxious heat. B: deep units. Left and right sides show averaged PSTHs of 15 deep dorsal horn units to ic histamine and noxious heat, respectively (format as in A). Inset shows locations of deep dorsal horn recording sites. WDR, wide dynamic range; HT, high threshold.


                              
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Table 1. Magnitude and decay of histamine- and noxious heat-evoked responses of superficial versus deep dorsal horn neurons

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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Fig. 5. Superficial WDR neuron isolated using histamine search stimulus. A: PSTH (binwidth: 1 s) of prolonged discharge to ic histamine (at arrow). Top inset shows mechanosensitive receptive field (black area) and ic injection site (arrow). B: same unit's response to heat (left PSTH; bar = 5-s stimulus) and repeat ic histamine (right PSTH). Application of heat caused a reduction in histamine-evoked discharge. C: same unit's responses to ic capsaicin, topical mustard oil, and ic nicotine (left to right PSTHs). D: recording site (top) and example of spike waveform (bottom).

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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Table 2. Responses of individual superficial dorsal horn units to different chemicals

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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Fig. 6. Example of effects of intrathecal (IT) morphine on responses of superficial WDR dorsal horn neuron to ic histamine and noxious heat. A: PSTHs of responses to 3% ic histamine, before morphine (left PSTH), 30 min following 3 indicated doses of IT morphine (2nd, 3rd, and 4th PSTHs from left), and following IT naloxone (right PSTH). Left inset shows mechanosensitive receptive field (black area) and histamine injection site (arrow). Right inset shows example of spike waveform. B: PSTHs of same unit's responses to noxious heat at the indicated times relative to IT morphine and naloxone. Note facilitation of histamine responses at low morphine doses, inhibition at the highest (10 µM) dose, and naloxone reversal.

The graphs in Fig. 7 plot individual (thin lines) and mean (thick line with error bars) unit responses at selected time points following IT administration of different concentrations of morphine and are grouped according to the effect of low concentrations of morphine. Superficial units whose histamine responses were facilitated at low morphine concentrations are shown in Fig. 7A, and units whose histamine responses were suppressed are shown in Fig. 7B. Morphine effects, whether facilitatory or inhibitory, were concentration dependent (Fig. 7, A and B). The degree of morphine-induced facilitation was significantly greater than morphine-induced suppression at both 100-nM and 1-µM concentrations (P < 0.0001, unpaired t-test) even when including the four units unaffected by morphine in the analysis. In five of nine units morphine facilitated both histamine- and heat-evoked responses, while in four units morphine facilitated histamine-evoked responses but suppressed heat-evoked responses.



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Fig. 7. Effects of morphine on dorsal horn unit responses to ic histamine and noxious heat (52°C/5 s). Units were grouped according to whether low-dose morphine (0.1-1 µM) facilitated responses to histamine (A) or heat (C), or suppressed responses to histamine (B) or heat (D). Each graph plots unit responses to the histamine or heat stimulus before (Pre), and following IT administration of morphine and naloxone at the indicated doses and times postapplication (time indicated in parentheses below, in min). Thin lines: individual units. Dashed lines connect each unit's response following morphine to its response following naloxone. Thick black line with error bars (SE): mean response. * Significantly different from premorphine control (P < 0.05, paired t-test). A: facilitation of ic histamine-evoked responses by low-dose morphine. B: suppression of ic histamine-evoked responses by low-dose morphine. C: facilitation of noxious heat-evoked responses by low-dose morphine. D: suppression of noxious heat-evoked responses by low-dose morphine. In 3/6 cases morphine suppression was clearly reversed by naloxone. E and F: graphs as in C and D for deep dorsal horn units.

Morphine (100 nM, 1 µM) facilitated (>130%) noxious heat-evoked responses in 15/25 of superficial units (Fig. 7C), reduced responses (below 70%) in 8/25 units (Fig. 7D), and had no effect in 2. Again, the facilitatory or inhibitory effects of morphine were concentration dependent. The magnitude of morphine-induced facilitation of heat-evoked responses was significantly greater than that of morphine-induced suppression at both 100-nM and 1-µM concentrations (P < 0.01, unpaired t-test) even when including the units unaffected by morphine. In five units morphine facilitated responses to both noxious heat and histamine (Fig. 6), while in eight units morphine facilitated heat-evoked responses but suppressed histamine-evoked responses. 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.

A higher concentration of morphine (10 µM), when tested, suppressed the responses of most superficial units to both histamine and noxious heat (Figs. 6 and 7, A and C). The 10 µM morphine depressed unit responses to histamine and noxious heat to a degree that was significantly below the same units' responses to heat and histamine recorded previously after application of 100 nM or 1 µM morphine (P < 0.05, paired t-test).

We investigated whether IT naloxone reversed the effects of IT morphine on superficial unit responses to histamine or noxious heat. In Fig. 7, the dashed lines connect individual unit responses to histamine (Fig. 7, A and B) or noxious heat (Fig. 7, C and D) following low-concentration morphine with the same unit's response following IT naloxone. For histamine-evoked responses, naloxone appeared to reverse morphine-induced facilitation in two of five cases (Fig. 7A), and suppression in five of eight cases (Fig. 7B). For noxious heat-evoked responses, naloxone reversed morphine-induced facilitation in two of five cases (Fig. 7C) and suppression in three of five cases (Fig. 7D).

Overall, naloxone significantly reversed the depressant (but not facilitatory) effect of low-concentration morphine. Thus responses to histamine and heat were significantly greater at 30 min postnaloxone than they were following 1 µM morphine (P < 0.05, paired t-test), and were not significantly different from their respective premorphine baseline responses (P > 0.05, paired t-test).

In a smaller number of units, we tested the effect of naloxone on the depressant effect of the higher (10 µM) morphine concentration; these cases are indicated by the solid lines connecting data points at 10 µM morphine with naloxone in Fig. 7, A-D. Naloxone reversed morphine-induced inhibition of histamine-evoked responses in two of four cases (Fig. 7, A and B) and of heat-evoked responses in four of four cases (Fig. 7, C and D).

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.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    ACKNOWLEDGMENTS

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.


    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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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