Departments of 1Neuroscience and 2Psychology, Schrier Research Laboratory, Brown University, Providence, Rhode Island 02912
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ABSTRACT |
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Strangman, Nicole M. and J. Michael Walker. Cannabinoid WIN 55,212-2 Inhibits the Activity-Dependent Facilitation of Spinal Nociceptive Responses. J. Neurophysiol. 82: 472-477, 1999. Cannabinoids suppress nociceptive processing of acute stimuli, but little is known about their effects on processes that lead to hyperexcitability of nociceptive neurons following prolonged noxious stimulation. Wind-up, the increasingly strong response of spinal nociceptive neurons to repetitive noxious electrical stimuli, results from a fast-rising cumulative depolarization and increase in intracellular calcium concentration. These processes produce central sensitization, the increased excitability of spinal nociceptive neurons that contributes to the hyperalgesia and allodynia associated with chronic pain. Intravenous injection of the potent, synthetic cannabinoid agonist WIN 55, 212-2, but not the inactive enantiomer, WIN 55,212-3, dose-dependently decreased the wind-up of spinal wide dynamic range and nociceptive-specific neurons independent of acute responses to activation of low- and high-threshold primary afferents. This is the first direct evidence that cannabinoids inhibit the activity-dependent facilitation of spinal nociceptive responses.
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INTRODUCTION |
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Cannabinoids dampen the pain behavior evoked by a
variety of different acute, noxious stimuli (Buxbaum
1972; Martin et al. 1993
; Sofia et al.
1973
). Consistent with these behavioral findings, cannabinoids
inhibit the responses of nociceptive neurons in the spinal dorsal horn
and ventral posterolateral nucleus of the thalamus to acute noxious
pressure and heat (Hohmann et al. 1995
, 1999
; Martin et al. 1996
). Together, these data provide strong
evidence that cannabinoids disrupt the transmission of brief,
nociceptive stimuli that lead to acute pain.
Less well characterized are the effects of cannabinoids on reactions to
prolonged nociceptive stimulation. Whether cannabinoids modulate
responses to prolonged noxious stimuli is a question of great
importance because chronic pain involves mechanisms additional to those
involved in acute pain. Kosersky et al. (1973) found that
9-tetrahydrocannabinol (
9-THC)
raised the threshold for pressure-induced vocalization in rats whose
hindpaws had been inflamed by yeast or carrageenin. Moss and
Johnson (1980)
and Tsou et al. (1996)
reported
that behavioral responses to a noxious chemical stimulus in the
formalin test were significantly reduced by
9-THC and
WIN 55,212-2, respectively. In the latter study, the cannabinoid
markedly attenuated formalin-induced c-fos expression in the
spinal cord as well. More recently, Herzberg et al.
(1997)
reported that intraperitoneal injection of WIN 55, 212-2 decreased thermal hyperalgesia and mechanical allodynia in rats with
chronic constriction injury of the sciatic nerve. These findings
suggest that cannabinoids suppress responses to prolonged noxious
stimulation as effectively as they do responses to acute noxious
stimulation. However, the mechanisms by which cannabinoids produce
these effects are unknown.
Cutaneous tissue injury leads to a constellation of changes in spinal
excitability, which includes elevated spontaneous firing, increased
response amplitude and duration, decreased threshold, enhanced
afterdischarge to repeated stimuli, and expanded receptive fields
(Woolf 1983). The persistence of these changes, which
are collectively termed central sensitization, appears to be
fundamental to the prolonged enhancement of pain sensitivity definitive
of chronic pain. Physiological changes similar to those associated with
central sensitization are produced in spinal nociceptive neurons by
repetitive, low-frequency, noxious electrical stimulation of C-fibers
(Cook et al. 1987
; Ren et al. 1992
;
Wall and Woolf 1986
; Woolf and Wall
1986
). This type of stimulation also produces wind-up, a
centrally mediated increase in both the frequency and duration of
spinal nociceptive responses (Mendell 1966
). Wind-up and
central sensitization exhibit similar pharmacological susceptibilities (Woolf and Thompson 1991
; Xu et al.
1992
), a reflection of their common reliance on synaptic
processes that elevate levels of intracellular calcium
(Sivilotti et al. 1993
; Thompson et al.
1990
; Woolf et al. 1988
). As discussed by
Woolf (1996)
, it is not clear whether wind-up is an
essential process in the etiology of the hyperalgesia and allodynia
associated with chronic pain syndromes. However, the synaptic processes
that produce wind-up are sufficient to produce central sensitization,
which appears to be an important component of hyperalgesia and
allodynia. In this study, we examined the effect of the potent,
synthetic cannabinoid agonist WIN 55,212-2 on wind-up of spinal dorsal
horn neurons.
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METHODS |
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All protocols were reviewed and approved by the Brown University Institutional Animal Care and Use Committee.
Male Sprague-Dawley rats (Charles River, Boston, MA) were anesthetized
by urethan (25%) injection (1.5 g/kg ip) and mounted in a stereotaxic
frame. A laminectomy was performed at the
T12-L1 vertebrae, and the spinal cord was
immobilized with spinal clamps and immersed in warm mineral oil.
Extracellular, single-unit responses were recorded from 22 wide dynamic
range and 8 nociceptive-specific neurons in the lumbar spinal dorsal
horn using 5-M stainless steel electrodes. Units were characterized
based on the pattern of their responses to natural stimulation and the
latency of their responses to transcutaneous, C-fiber intensity
electrical stimulation applied to the receptive field.
The procedure of Chapman et al. (1994) was used to
elicit wind-up. After isolation and characterization of a cell, 16 transcutaneous, electrical stimuli (2 ms in duration, 2 s apart)
at 3 times the threshold of the C-fiber response (~3 mA) were applied
to the receptive field. After a baseline trial, WIN 55,212-2 [0.125, 0.25 or 0.5 mg/kg; dosages selected on the basis of work by
Hohmann et al. (1995)
and preliminary data], the
inactive enantiomer WIN 55,212-3 (0.5 mg/kg), or vehicle (5:5:90,
ethanol/emulphor/0.9% saline) was injected intravenously, and
subsequent trials were performed at 5-min intervals. Seven cells were
lost after 25-75 min of recording. All others were tested for a total
of 20 trials.
Unit responses to transcutaneous electrical stimulation were subdivided
into A- (0-20 ms), A
- (20-90 ms), and C- (90-800 ms)
fiber-mediated components. The acute response to high- or low-threshold primary afferent activation (the input) was calculated by
tallying the total number of A
-, A
-, or C-fiber latency action potentials that occurred in response to the first stimulus of a trial.
Wind-up was calculated as
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At the end of each experiment, the recording site was marked by iron
deposition (2 µA for 20-30 s) and perfusion with Prussian Blue
solution. Spinal cord sections were stained with Neutral Red, and
recording sites were determined microscopically using the stereotaxic
atlas of Paxinos and Watson (1986).
Scores were collapsed across postinjection trials 1-5, determined in
previous experiments by Hohmann et al. (1995) to
encompass the period of maximal effect of WIN 55,212-2. Drug effects on acute responses and wind-up were statistically compared using the
Student's t-test and the Bonferroni adjustment for multiple planned comparisons. P < 0.05 was considered
statistically significant. Multiple linear regression using the initial
A
-, A
-, and C-fiber latency responses as predictor variables was
used to examine the relationship between the acute response and wind-up
based on data from the control and 0.5 mg/kg WIN 55,212-2 groups.
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RESULTS |
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The composition of each group was as follows: the WIN 55,212-3 group was composed of two wide dynamic range neurons and two nociceptive-specific neurons; the WIN 55,212-2 0.125 mg/kg group was composed of four wide dynamic range and three nociceptive-specific neurons; the vehicle and WIN 55,212-2 0.25 mg/kg groups each contained six wide dynamic range neurons and one nociceptive-specific neuron; and the WIN 55,212-2 0.5 mg/kg group was composed of three wide dynamic range neurons and one nociceptive-specific neuron. One wide dynamic range neuron that showed erratic responses to stimulation was excluded as an outlier. Recording sites could be microscopically determined for 26 of 29 cells. In 25 cases the recording site was localized to the deep laminae III-V of the lumbar spinal dorsal horn. The two remaining recording sites were located in the most superficial laminae, I and II.
Wind-up stimulation produced a gradual increase in the frequency and
duration of the C-fiber discharge and afterdischarge of spinal
nociceptive neurons, an effect that was highly repeatable over time
(Fig. 1). Treatments with vehicle or the
cannabinoid inactive enantiomer WIN 55,212-3 were virtually identical
in their failure to produce a significant change in wind-up, the
C-fiber response, or the acute A response; therefore these two
groups were pooled into a single control group for all subsequent
analyses.
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Before drug injection, the control group did not differ from the WIN
55,212-2 groups in measures of wind-up or acute responses to A- or
C-fiber activation. WIN 55,212-2 0.25 mg/kg and WIN 55,212-2 0.5 mg/kg
significantly inhibited wind-up as compared with the control group
(Figs.
1-3,
F(1, 16) = 2.92, F(1, 13) = 3.78), whereas
treatment with WIN 55,212-2 0.125 mg/kg failed to produce any effects
on wind-up (Fig. 2). Treatment with WIN 55,212-2 at the dose of 0.5 mg/kg but not 0.125 mg/kg or 0.25 mg/kg significantly decreased the
acute C-fiber response as compared with control [Fig. 2,
F(1, 13) = 3.03]. However, all doses of WIN 55,212-2 failed
to alter the acute response to A
-fiber and A
-fiber activation.
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As noted above, WIN 55,212-2 suppressed acute C-fiber responses in
dorsal horn neurons when administered at the dose of 0.5 mg/kg. A more
detailed examination of the data revealed that even at this dose, it is
possible to dissociate the effects on the acute C-fiber response from
those on wind-up. Thus wind-up score and the initial C-fiber latency
response (but not the initial A or A
latency response) were
correlated (Fig. 3, r = 0.71, P < 0.0005), but they were frequently dissociated. For example, even with
an acute response of 0 action potentials, a neuron recorded in an
animal that received vehicle showed a wind-up response of 180, ranging
3-180 times higher than that of neurons recorded in animals that
received WIN 55,212-2 (0.5 mg/kg) and also showed zero acute response
to the stimulation. At identical levels of acute response, wind-up of
control cells was much greater than that of WIN 55,212-2 cells. In five
cases WIN 55,212-2 (0.5 mg/kg) cells did not show any wind-up, and in
three additional cases the wind-up score did not exceed four action
potentials. However, in all 9 cases where the acute C-fiber response of
vehicle and WIN 55,212-3 cells was equal to or less than that of WIN
55,212-2 (0.5 mg/kg) cells, these control cells showed wind-up of 36 action potentials or greater. The mean wind-up score for neurons that responded with two action potentials to the first stimulus was 40.3 ± 23.9 (mean ± SE) for WIN 55,212-2 (0.5 mg/kg) cells (n = 4) and 123.3 ± 43.7 for vehicle
cells (n = 3). Taken together, these observations
indicate that the suppression of the acute C-fiber response by the
highest dose of the cannabinoid agonist can be dissociated from its
effect on wind-up.
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DISCUSSION |
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The cannabinoid agonist WIN 55,212-2 inhibited the wind-up of spinal wide dynamic range and nociceptive-specific neurons in response to repeated noxious stimulation. The dose dependency of this effect and the lack of efficacy of the inactive enantiomer WIN 55,212-3 indicate that the suppression was mediated by cannabinoid receptors. The inhibition of wind-up was independent of any reduction in the acute response to C-fiber activation. At the dose of 0.25 mg/kg, WIN 55,212-2 suppressed wind-up without altering the acute C-fiber response. Although the highest dose of WIN 55,212-2 (0.5 mg/kg) suppressed both wind-up and the acute response to C-fiber activation, close examination of the data revealed a dissociation between these two measures. These findings are consistent with an action of the cannabinoid on synaptic processes that produce increased spinal excitability upon repeated C-fiber activation. This study thus provides evidence that cannabinoids suppress the facilitation of spinal responses following repeated noxious stimulation independent of acute responses to low- and high-threshold primary afferents.
The effect of WIN 55,212-2 is distinct from that of morphine, which
inhibits wind-up only at doses that also suppress the acute response to
C-fiber activation (Dickenson and Sullivan 1986), suggesting that opiates may act relatively selectively to suppress acute nociceptive transmission but making it difficult to judge, based
on grouped data, whether there is an independent effect on the synaptic
processes that lead to wind-up. We suggest that a cell-by-cell
comparison of the acute C-fiber response and wind-up may be useful in
cases such as these for teasing out a drug's relative effects on the
acute responses and wind-up. The ability of WIN 55,212-2 to suppress
the repetitive stimulus-induced facilitation of spinal excitability,
independent of acute responses, may account in part for the higher
potency of cannabinoids in behavioral models of chronic pain
(Herzberg et al. 1997
).
The failure of WIN 55,212-2 to suppress the responses of spinal wide
dynamic range neurons to A-fiber activation is consistent with the
drug's lack of effect on responses of nonnociceptive neurons in the
spinal cord and thalamus to nonnoxious stimulation (Hohmann et
al. 1995
; Martin et al. 1996
). This observation
provides a further indication that cannabinoids produce analgesia
rather than anesthesia. The lack of effect of WIN 55,212-2 on acute
responses to A
-fiber activation may seem surprising in light of the
profound suppression by this drug of the responses of nociceptive
neurons to acute noxious heat and pressure (Hohmann et al. 1995
,
1999
; Martin et al. 1996
). However, this failure
to detect an effect of WIN 55,212-2 on acute responses to A
-fiber
activation is likely due to a floor effect pursuant to the low
frequency of the A
response.
Spinal transection profoundly attenuates the suppression of
noxious-heat evoked activity in spinal wide dynamic range neurons by
systemically administered WIN 55,212-2 (Hohmann et al. 1996, 1999
). Suppression of spinal nociceptive responses can also be produced by intraventricular administration of WIN 55,212-2 (Hohmann and Walker 1999
). Together with the behavioral
findings that cannabinoids microinjected into the periaqueductal gray
and dorsal raphe inhibit the tail-flick reflex (Martin et al.
1995
), these data provide strong evidence that cannabinoids act
supraspinally to modulate nociceptive processing. However, Hohmann
identified a subpopulation of neurons in spinal rats that exhibit
moderate suppression following intravenous administration of WIN
55,212-2, and Lichtman and Martin (1991)
reported modest
inhibition by a cannabinoid of thermal pain sensitivity in spinal
animals. Furthermore, topical administration of cannabinoids onto the
spinal cord inhibits spinal nociceptive responses (Hohmann et
al. 1998
). Thus the actions of cannabinoids on a subpopulation
of spinal nociceptive neurons may also contribute to the dampening of
nociceptive transmission and pain. Recently, cannabinoid (CB1) receptor
mRNA was detected in substance P containing neurons in the dorsal root
ganglia (Hohmann and Herkenham 1998
, 1999
). Consistent
with the localization of CB1 receptors to the cell bodies of primary
afferent neurons, a dose of anandamide that lacked analgesic efficacy
when administered systemically, inhibited the development of
carrageenan-induced thermal hyperalgesia when administered peripherally
(Richardson et al. 1998
). These data raise the
possibility that cannabinoids might modulate nociception by decreasing
neurotransmitter release from primary afferents. Because WIN 55,212-2 was delivered by intravenous route in the present study, we cannot
ascertain whether the drug's site of action was spinal, supraspinal,
peripheral, or some combination of the three. Further work on this
question would undoubtedly provide important insights into the
mechanisms of cannabinoid analgesia.
The role of wind-up in the development of central sensitization and the
consequent hyperalgesia and allodynia in chronic pain is uncertain.
Wind-up relies on the production of a fast-rising cumulative
depolarization that increases the level of intracellular calcium
(Sivilotti et al. 1993; Thompson et al.
1990
, 1993
; Urban and Randic
1984
; Woolf et al. 1988
). Likewise, experimental
manipulations that increase intracellular calcium produce central
sensitization (Woolf and Wiesenfeld-Hallin 1986
).
However, central sensitization may occur in the absence of action
potential production in spinal nociceptive neurons (Liu and
Sandkühler 1997
; Magerl et al. 1998
). For
example, depolarizations subthreshold for wind-up can produce heterosynaptic facilitation, a phenomenon analogous to central sensitization (Thompson et al. 1993
). Therefore, as
discussed by Woolf (1996)
, it is not clear that wind-up
is necessary for central sensitization, but it is clear that wind-up
stimulation activates synaptic processes that are sufficient to produce
central sensitization (Cook et al. 1987
; Ren et
al. 1992
; Wall and Woolf 1986
; Woolf and
Wall 1986
).
In light of the above, it is notable that the suppression of wind-up by
WIN 55,212-2 is likely due to inhibition of calcium entry into spinal
neurons. Cannabinoids interfere with several components of the calcium
signal cascade including the influx of calcium through N- and Q-type
Ca2+ channels (Felder et al. 1993,
1995
; Mackie and Hille 1992
), the release
of calcium from intracellular stores (Filipeanu et al. 1997
), and the activation of protein kinase C (De
Petrocellis et al. 1995
; Hillard and Auchampach
1994
). The central cannabinoid receptor is also coupled to the
Gi protein, and inhibition of adenylate cyclase might also
underlie this effect (Howlett 1985
; Howlett et
al. 1987
).
If the suppression of wind-up by cannabinoids is due to inhibition of
calcium entry, then one would expect cannabinoids to be effective
inhibitors of central sensitization produced by a variety of
mechanisms. In this regard, it is relevant to note that cannabinoids
eliminated hyperalgesia and allodynia in the Bennett model of
neuropathic pain at doses that produced no observable side effects
(Herzberg et al. 1997). In fact, the potency of WIN 55,212-2 in this study was markedly higher than in models of acute pain. Likewise, cannabinoids inhibit hyperalgesia due to inflammation (Kosersky et al. 1973
; Moss and Johnson
1980
; Richardson et al. 1998
; Sofia et
al. 1973
; Tsou et al. 1996
). The observations
that cannabinoids inhibit calcium entry, wind-up, and the allodynia and
hyperalgesia following nerve injury or inflammation, suggest the
possibility that cannabinoids may act as general inhibitors of central
sensitization by inhibiting calcium entry. Studies aimed at determining
the effects of cannabinoids on intracellular calcium following
inflammation and nerve injury are needed to examine this possibility.
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ACKNOWLEDGMENTS |
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Financial support for this work was provided by the National Institutes of Health Grants K02MH-01083, NS-33247, DA-10043, and DA-10536.
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FOOTNOTES |
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Address for reprint requests: J. M. Walker, Dept. of Psychology, Brown University, P.O. Box 185389, Waterman St., Providence, RI.
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 16 December 1998; accepted in final form 4 March 1999.
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REFERENCES |
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