Department of Neurological Surgery, Oregon Health Sciences University, Portland, Oregon 97201
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Heinricher, M. M., S. McGaraughty, and V. Tortorici. Circuitry Underlying Antiopioid Actions of Cholecystokinin Within the Rostral Ventromedial Medulla. J. Neurophysiol. 85: 280-286, 2001. It is now well established that the analgesic actions of opioids can be modified by "anti-analgesic" or "antiopioid" peptides, among them cholecystokinin (CCK). Although the focus of much recent work concerned with CCK-opioid interactions has been at the level of the spinal cord, CCK also acts within the brain to modify opioid analgesia. The aim of the present study was to characterize the actions of CCK in a brain region in which the circuitry mediating the analgesic actions of opioids is relatively well understood, the rostral ventromedial medulla (RVM). Single-cell recording was combined with local infusion of CCK in the RVM and systemic administration of morphine in lightly anesthetized rats. The tail-flick reflex was used as a behavioral index of nociceptive responsiveness. Two classes of RVM neurons with distinct responses to opioids have been identified. OFF cells are activated, indirectly, by morphine and µ-opioid agonists, and there is strong evidence that this activation is crucial to opioid antinociception. ON cells, thought to facilitate nociception, are directly inhibited by opioids. Cells of a third class, NEUTRAL cells, do not respond to opioids, and whether they have any role in nociceptive modulation is unknown. CCK microinjected into the RVM by itself had no effect on tail flick latency or the firing of any cell class but significantly attenuated opioid activation of OFF cells and inhibition of the tail flick. Opioid suppression of ON-cell firing was not significantly altered by CCK. Thus CCK acting within the RVM attenuates the analgesic effect of systemically administered morphine by preventing activation of the putative pain inhibiting output neurons of the RVM, the OFF cells. CCK thus differs from another antiopioid peptide, orphanin FQ/nociceptin, which interferes with opioid analgesia by potently suppressing all OFF-cell firing.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
It is now well established that
the analgesic actions of opioids can be modified by endogenous
"antiopioid" peptides, and the role of cholecystokinin (CCK) as a
physiological opioid antagonist has received particular attention.
Administration of CCK agonists can diminish opioid analgesia
(Faris et al. 1983; Itoh et al. 1982
;
Li and Han 1989
), whereas CCK antagonists enhance the
antinociceptive effects of exogenous and endogenous opioids and can
slow or prevent the development of opioid tolerance in some paradigms
(Chapman et al. 1995
; Dourish et al.
1990
; Hoffmann and Wiesenfeld-Hallin 1994
;
Maldonado et al. 1993
, 1995
; Mitchell et al.
2000
; Noble et al. 1995
; Rezayat et al.
1994
; Valverde et al. 1994
; Watkins et
al. 1984
, 1985a
,b
; Wiesenfeld-Hallin et al.
1990
; Zhou et al. 1993
). CCK is also implicated
in environmentally induced changes in nociception (Chen et al.
1998
; Hendrie et al. 1989
; Lavigne et al.
1992
; Wiertelak et al. 1992
, 1994
). In addition,
there is evidence that alterations in the availability of CCK
contribute to the changes in analgesic potency of opioids seen in
conditions of inflammation or stress and some pathological pain states
(Coudore-Civiale et al. 2000
; Friedrich and
Gebhart 2000
; Hawranko and Smith 1999
; Nichols et al. 1995
, 1996
; Stanfa and Dickenson
1993
, 1994
; Wiesenfeld-Hallin et al. 1999
).
The focus of much of the recent work concerned with CCK-opioid
interactions has been at the level of the spinal cord (Stanfa et
al. 1994; Wiesenfeld-Hallin et al. 1999
), and
although CCK has also been shown in behavioral studies to act within
the brain to inhibit opioid analgesia (Kovelowski et al.
2000
; Li and Han 1989
; Mitchell et al.
1998
; Noble et al. 1993
; Pu et al.
1994
; Vanderah et al. 1996
; Watkins et
al. 1985b
), the neural circuitry through which this occurs has
not been identified. The goal of the present experiments was to
determine how CCK influences opioid effects on the circuitry within the
rostral ventromedial medulla (RVM), a region with a well-documented
role in opioid analgesia (Basbaum and Fields 1984
;
Fields et al. 1991
; Heinricher and Morgan 1999
).
Three physiologically distinct populations of neurons have been
identified in the RVM. Only one group of RVM neurons, OFF cells, is activated by µ-opioid agonists (Fields et al.
1983b; Heinricher et al. 1994
). We have shown
that this activation is indirect (Heinricher et al.
1992
) and sufficient to produce behaviorally measurable
antinociception (Heinricher and Tortorici 1994
;
Heinricher et al. 1994
). Cells of a second class,
ON cells, display a sudden increase in activity beginning
just before the occurrence of nocifensive reflexes, and likely exert a
permissive or even facilitating effect on nociception.
ON-cell firing is directly inhibited by µ-opioid agonists
(Bederson et al. 1990
; Fields 1992
;
Heinricher et al. 1992
; Pan et al. 2000
).
NEUTRAL cells show no change in activity associated with
nociceptive responses and do not respond to opioids given by any route
(Barbaro et al. 1986
; Gao et al. 1998
).
Their role, if any, in nociceptive modulation remains unclear. The
advantages to using the RVM for this analysis of CCK-opioid
interactions are thus that the responses of RVM neurons to opioid
administration are known and that how each cell class contributes to
opioid analgesia is relatively well understood.
CCK has been shown to have excitatory effects in a number of brain
regions, including the hippocampus, nucleus tractus solitarius, dorsal
horn, and periaqueductal gray, although inhibitory effects are
sometimes seen (Albrecht et al. 1994; Boden and
Woodruff 1994
; Jeftinija et al. 1981
; Liu
et al. 1994
; Miller et al. 1997
). Thus a
reasonable hypothesis would be that CCK acting in the RVM could disrupt
morphine antinociception by activating ON cells, blocking opioid inhibition of these neurons. We tested this possibility by
examining the effects of locally applied CCK on the activity of RVM
neurons and their responses to systemically administered morphine. We
found that, contrary to expectation, CCK attenuated the analgesic
actions of systemically administered morphine by interfering with
opioid activation of OFF cells.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Animals and surgical preparation
All experimental procedures were approved by the Institutional Animal Care and Use Committee at Oregon Health Sciences University. Male Sprague-Dawley rats (250-300 g, B and K, Hayward, CA) were anesthetized with pentobarbital (60 mg/kg ip), and catheters were inserted into the external jugular veins for administration of anesthetic, morphine, and naloxone. The rat was placed in a stereotaxic apparatus, a hole was drilled in the skull over the cerebellum, and the dura was removed to allow placement of an electrode/microinjection cannula assembly in the medulla. Body temperature was maintained at approximately 37°C by a circulating-water pad.
Following surgery, the anesthetic level was allowed to lighten until
the tail-flick response (TF), a spinal nociceptive reflex, could be
elicited by application of noxious heat using a feedback-controlled projector lamp focused on the blackened ventral surface of the tail.
The animals were then maintained in a lightly anesthetized state with a
continuous infusion of methohexital (15-30 mg · kg1 · h
1 iv) as
previously described (Barbaro et al. 1989
).
Nociceptive testing
TF latency was used as a measure of nociceptive responsiveness. The heat was applied to spots 2, 3, or 4 cm from the tip of the tail in succession. Each trial consisted of a linear increase in temperature at approximately 1.8°C/s from a holding temperature of 34°C until the TF occurred or to a maximum of 52°C at 10.6 s. TF trials were carried out at 5-min intervals throughout the experiment.
Recording and drug administration
A gold- and platinum-plated stainless steel recording microelectrode (Frederick Haer, Brunswick, ME) was glued parallel to a single-barrel glass infusion pipette with an outer diameter of 75-80 µm in such a way that the tips were separated laterally by 100-300 µm. This separation allowed us to maintain well-isolated recordings of single neurons during and after infusions of 200 nl of saline vehicle or drug solution in the RVM in a reasonable number of experiments. The assembly was oriented in the electrode carrier so that the assembly straddled the midline, with both electrode and infusion pipette within the RVM. The infusion pipette was attached to a 1 µl Hamilton syringe with a length of PE-50 tubing for drug infusion.
RVM neurons were classified as previously described (Fields et
al. 1983a). Spike waveforms were monitored and stored for
off-line analysis (Datawave Systems, Thornton, CO) to ensure that the
unit under study was unambiguously discriminated throughout the
experiment and to verify that the solutions applied did not have local
anesthetic effects. Spike times were stored with a temporal resolution
of 0.1 ms. OFF cells were characterized by an abrupt pause
in ongoing activity beginning just prior to the occurrence of the TF.
ON cells were identified by a sudden burst of activity
beginning just prior to the occurrence of the TF. Cells of a third
class, neutral cells, were identified by no change in activity
associated with the tail flick or with noxious or innocuous somatic stimulation.
Protocol and data analysis
One set of experiments examined the effect of systemic
administration of CCK on the ability of systemically administered
morphine to alter TF latency and RVM neuronal firing. Following three
baseline TF trials, CCK-8S (Tocris Cookson, CCK), the predominant CCK
peptide in the CNS (Rehfeld et al. 1985), was given
intravenously (4 µg/kg). This was followed 10 min (2 TF trials) later
by systemic administration of morphine sulfate (MS) (0.375-1.5 mg/kg
iv). TF latency and cell activity were then monitored for an additional
40 min.
In a second group of animals, we determined the effect of CCK microinjection into the RVM on TF latency and the ongoing and TF-related discharges of RVM neurons. Following three baseline TF trials, CCK (10 ng) was infused into the RVM. This dose was chosen because pilot studies had shown that microinjection of 10 ng CCK into the RVM had an effect on morphine antinociception that was similar to that of 4 µg given intravenously. All RVM injections were made in a volume of 200 nl over a period of approximately 4 min. TF latency and cell activity were then monitored for a period of 45 min.
A third set of experiments used a similar paradigm to investigate the effect of CCK microinjection into the RVM on the actions of systemically administered morphine. Following three baseline TF trials, CCK (10 ng) or saline vehicle was infused into the RVM. RVM infusions were followed 10 min (2 TF trials) later by systemic administration of morphine sulfate (0.375-1.5 mg/kg iv). TF latency and cell activity were then monitored for an additional 40 min.
Only one protocol was performed in each of the 249 animals used in these experiments. Data are presented only from experiments in which the neuron was successfully held throughout the entire testing period.
TF latencies and cell parameters obtained in the baseline period were
compared with the average of the post-CCK or the 15- through 30-min
postmorphine time points. Inhibition of the TF was quantified as
percent maximum possible effect, i.e., %MPE = 100*(postdrug
latency - baseline latency)/(10.6 - baseline latency). Three cell
parameters were analyzed. 1) Ongoing activity. Because OFF and ON cells often show irregular
alternations between periods of silence and activity, cell activity
integrated over the 30 s prior to each TF trial was used as an
overall index of ongoing firing. 2) The OFF-cell
pause. The TF-related OFF-cell pause in firing was analyzed
for those tail flick trials on which the OFF cell was
active immediately prior to heat onset. TF-related inhibition of firing
was calculated by expressing the firing rate measured in the 2-s period
beginning 1 s before the TF (or, if no TF occurred following
morphine administration, the mean TF latency in baseline) as a
percentage of that in the 5-s epoch immediately prior to heat onset. A
value of 100% would thus indicate no slowing associated with tail
heating/flick, a value of 0% complete inhibition for at least 2 s. These intervals were chosen based on the previously described
spontaneous firing pattern of RVM neurons and duration of the
OFF-cell pause (Barbaro et al. 1989).
3) ON-cell TF-related burst. Total spike count
in the 3-s period beginning 1 s before the TF was recorded for all
TF trials.
Data are presented as means ± SE. Wilcoxon's signed ranks test and Mann-Whitney U (for TF latencies), ANOVA and Student's t-test (for cell parameters) were used for statistical analysis of results; P < 0.05 was considered significant.
Histology
At the conclusion of the experiment, recording/infusion sites
were marked with an electrolytic lesion. Animals were killed with an
overdose of methohexital and perfused intracardially with physiological
saline followed by 10% formalin. Recording/infusion sites were
histologically verified and plotted on standardized sections
(Paxinos and Watson 1997). The distribution of sites was
within the RVM, as previously defined, comprising the nucleus raphe
magnus as well as the laterally adjacent reticular formation at the
level of the facial nucleus, including the nucleus reticularis gigantocellularis pars alpha and nucleus
paragigantocellularis lateralis.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Initial experiments were designed to identify a dose and protocol for CCK attenuation of morphine antinociception. TF latencies of the RVM-saline-injected, intravenous-saline, and no-treatment control groups did not differ (1-way ANOVA), and these three groups were combined for comparison with animals that received CCK. There was no difference in baseline TF latencies among the control or CCK groups (mean ± SE: 4.73 ± 0.08 s). As shown in Fig. 1, morphine given intravenously produced a dose-related increase in TF latency in the control animals, and this was significantly attenuated by administration of 4 µg/kg CCK intravenously 10 min prior to the morphine infusion. CCK given after morphine administration did not reverse morphine antinociception (data not shown).
|
We then examined the effects of CCK microinjected into the RVM on TF latency and RVM neuronal activity. As with systemic administration, microinjection of CCK into the RVM significantly attenuated the antinociceptive effect of systemically administered morphine (Fig. 1). CCK (10 ng) microinjected dorsal to the RVM did not alter the antinociceptive effect of morphine (%MPE for 0.75 mg/kg MS: 86.0 ± 12.6%, not significantly different from that of control animals, n = 4). Moreover, as shown in Fig. 2, CCK microinjection by itself had no effect on TF latency or the ongoing activity of ON, OFF, or NEUTRAL cells. In addition, the TF-related ON-cell burst was not altered (113 ± 14% of pre-CCK baseline) nor was the TF-related off-cell pause (TF-related inhibition was 116 ± 11% of that during baseline).
|
Analysis of morphine-induced changes in ON- and OFF-cell firing indicated that the ability of morphine to activate OFF cells was significantly reduced by CCK microinjected into the RVM. An example of two OFF cells recorded during administration of morphine is shown in Fig. 3. Morphine failed to produce a strong activation of the OFF cell recorded in the animal in which CCK was microinjected into the RVM, whereas that recorded following RVM saline showed a pronounced increase in activity and elimination of the OFF-cell pause. Quantitative analysis of group data demonstrates that the ongoing activity of OFF cells showed a significant increase in control animals, whereas the small increase in CCK-treated animals was not significant (Fig. 4A). The TF-related pause was also attenuated by morphine in a dose-related fashion in control animals (Fig. 4B) but not altered in CCK-treated animals, except at the highest morphine dose tested (1.5 mg/kg MS).
|
|
In contrast to the attenuation of morphine activation of OFF cells, CCK did not significantly alter the responses of ON cells to morphine administration. As shown in the examples in Fig. 5, ongoing activity was depressed by morphine in both CCK-treated and control animals with no significant difference between the CCK-treated and control groups (Fig. 6). Analysis of the TF-related burst was less clear-cut because of the wide variation in the amplitude of the burst in baseline, but as shown in Fig. 6 both CCK-treated and control groups displayed a similar trend of a dose-related decrease in the number of spikes associated with the TF (or at the mean time of the baseline TF in those animals in which the TF was inhibited following morphine).
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
CCK is found in neuronal processes throughout the RVM, where its
distribution overlaps that of enkephalin (Skinner et al. 1997). This peptide has been shown to have excitatory effects in a number of brain regions (Boden and Woodruff 1994
).
Since morphine and µ-opioid agonists depress ON-cell
firing but increase that of OFF cells, it seemed that CCK
applied within the RVM might interfere with the analgesic effects of
systemically administered morphine and that the mechanism would involve
an activation of ON cells that would counteract opioid
inhibition of these neurons. However, the suppression of
ON-cell firing by morphine was not significantly attenuated
by microinjection of CCK into the RVM, although the analgesic actions
of the opiate were significantly reduced. Rather the peptide prevented
opioid activation of OFF cells at a dose that had no effect
by itself on the ongoing firing of the neuron.
Our findings thus add further weight to existing evidence indicating
that the RVM is necessary for the analgesic actions of systemically
administered morphine and demonstrate conclusively that the relevant
opioid effect is activation of OFF cells (Heinricher et al. 1999; Mitchell et al. 1998
;
Proudfit 1980a
,b
; Valverde et al. 1996
;
Yaksh et al. 1977
; Young et al. 1984
).
Moreover the fact that CCK prevents opioid activation of
OFF cells without affecting inhibition of ON
cells has important implications for the organization of the pain
modulating circuitry within the RVM. Opioids do not activate
OFF cells directly but via disinhibition (Heinricher
et al. 1992
; Pan et al. 1990
). Previous models
of RVM circuitry have postulated that at least some ON
cells, which are directly responsive to µ-opioid agonists, are
GABAergic inhibitory interneurons. In this view, inhibition of
ON cells by morphine is responsible for disinhibition of
OFF cells, which in turn produces antinociception
(Fields et al. 1991
; Heinricher et al.
1992
). The present data fail to support this model insofar as
OFF- and ON-cell responses to opioids were
differentially modulated by CCK; OFF cells did not show a
significant morphine-induced increase in activity even though
ON-cell discharge was significantly depressed by the
opiate. These findings are thus compatible with a more recent proposal
that ON cells do not function as interneurons in RVM and
that opioid-sensitive GABAergic inputs from some site outside of the
RVM mediate the OFF-cell pause. µ-Opioid receptors are
thus presumably located presynaptically, inhibiting release of GABA
from the terminal (Heinricher et al. 1999
). Since
CCK by itself did not alter the firing of OFF cells (or any
RVM cell class), it is possible that the target for CCK is that same
afferent terminal. Alternatively, CCK could interfere with the ability of the OFF cell to respond to disinhibition, for example,
by reducing recruitment of endogenous
-opioid systems
(Hirakawa et al. 1999
; Kiefel et al.
1993
; Nichols et al. 1995
; Vanderah et
al. 1996
; Watkins et al. 1985a
) or with the
ability of OFF cells to respond to excitatory inputs
(Heinricher et al. 1999
). Distinguishing among these
possibilities will require further analysis at the cellular level.
Interestingly, Kovelowski et al. (2000) recently showed
that focal application of CCK within the RVM produced mechanical
allodynia and thermal hyperalgesia. The dose employed was six times
that used here, and CCK has been shown in electrophysiological studies to modulate effects of dopamine and opioids on neuronal discharge at
concentrations that by themselves have no effect with activation of the
target neuron only at higher concentrations (Crawley
1991
). This raises the possibility that a higher concentration
of CCK would activate ON cells and that this would explain
the hyperalgesia seen by Kovelowski et al. (2000)
.
Again, further studies will be required to examine this issue.
A number of neuropeptides, among them CCK, neurotensin, dynorphin, and
orphanin FQ/nociceptin, are now thought to have an antiopioid or
anti-analgesic effect by an action within the RVM (Heinricher et
al. 1997; Pan et al. 1997
; Urban and
Smith 1993
). The underlying mechanisms have been elucidated at
the circuitry level only for orphanin FQ/nociceptin, which profoundly
inhibits all three classes of RVM neurons, thus blocking opioid
activation of OFF cells and interfering with opioid
antinociception (Heinricher et al. 1997
). The mechanisms
through which orphanin FQ/nociceptin and CCK interfere with opioid
antinociception are thus quite different, although the ultimate target,
the OFF cells, is the same. Kappa agonists microinjected
into the RVM attenuate the antinociceptive action of µ-opioid
agonists microinjected into the periaqueductal gray.
Electrophysiological studies in vitro demonstrate that kappa receptor
activation inhibits a population of RVM neurons that are not directly
sensitive to opioids; these may be equivalent to OFF cells,
NEUTRAL cells, or both (Pan et al. 1997
).
This finding suggests that dynorphin and kappa receptor agonists
produce an anti-analgesic effect by inhibiting OFF cells,
thus preventing µ-receptor mediated disinhibition from leading to
activation. Whether this is the case, or whether there is an important
role for NEUTRAL cells, remains to be demonstrated in vivo.
Nevertheless it seems likely that dynorphin effects are more similar to
those of orphanin FQ/nociceptin in inhibiting OFF cells
than to those of CCK. Similarities and differences in the mechanisms
mediating the antiopioid effects of these peptides raise the
possibility that a range of neurotransmitters and neuropeptides are
brought into play to fine-tune pain modulating systems under different physiological conditions.
In conclusion, a number of investigators have obtained evidence that
endogenous CCK is increased in conditions of opioid tolerance and
certain neuropathic pain states marked by reduced opioid analgesic efficacy, and contributes to conditioned decreases in opioid analgesia (Idanpaan-Heikkila et al. 1997,b
; Nichols et al.
1995
, 1996
; Ossipov et al. 1994
; Stanfa
and Dickenson 1993
; Wiertelak et al. 1992
; Xu et al. 1994
). Our data suggest that the effects of
endogenous CCK on opioid analgesia may in part be due to the ability of
this peptide to regulate OFF-cell activation by these drugs.
![]() |
ACKNOWLEDGMENTS |
---|
This work was supported by grants from the National Institute on Drug Abuse (NIDA) (DA-05608) and the National Headache Foundation. S. McGaraughty was supported by a NIDA International Visiting Scientist and Technical Exchange Program fellowship and V. Tortorici by the Fogarty International Center.
![]() |
FOOTNOTES |
---|
Address for reprint requests: M. M. Heinricher, Dept. of Neurological Surgery, L-472, Oregon Health Sciences University, Portland, OR 97201 (E-mail: heinricm{at}ohsu.edu).
Received 11 July 2000; accepted in final form 21 September 2000.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|