Differential mediation of descending pain facilitation and inhibition by spinal 5HT-3 and 5HT-7 receptors

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DOI: 10.1016/j.brainres.2009.05.001 · Source: PubMed
Abstract
The rostroventromedial medulla (RVM) is an important source of descending modulatory systems that both inhibit and facilitate pain at the level of the spinal cord. Noxious stimuli can activate serotonergic neurons in the RVM and accelerate the turnover of 5-HT in the spinal cord. While numerous studies suggest a bidirectional role for serotonergic transmission at the spinal level, the subtypes of the 5-HT receptors that are associated with descending facilitation or inhibition have not been clearly determined. Here, we explore the relative contribution of spinal 5-HT7 and 5-HT3 receptors to antinociception or hyperalgesia associated with states of enhanced net descending inhibition or facilitation from the RVM. In uninjured rats, RVM microinjection of morphine produced dose-dependent antinociception in the noxious thermal paw flick test while RVM microinjection of CCK produced thermal hyperalgesia and tactile allodynia. Spinal administration of the 5-HT7 antagonist SB-269970, but not of the 5-HT3 antagonist ondansetron, blocked the antinociceptive effects of RVM morphine. In contrast, hyperalgesia induced by RVM-CCK was blocked by spinal ondansetron, but not by SB-269970. The antinociceptive effects of systemic morphine were also blocked by spinal SB-269970 but not ondansetron while hyperalgesia and allodynia resulting from SNL injury were blocked by spinal ondansetron, but not SB-269970. These studies suggest that descending pain inhibitory or facilitatory pathways from RVM act ultimately in the spinal cord in acute and chronic pain states through activation of 5-HT7 and 5-HT3 receptors, respectively.

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Research Report
Differential mediation of descending pain facilitation and
inhibition by spinal 5HT-3 and 5HT-7 receptors
Ahmet Dogrul
a
, Michael H. Ossipov
b,
, Frank Porreca
b
a
Gulhane Military Academy of Medicine, Department of Pharmacology, Ankara, 06018, Turkiye
b
University of Arizona College of Medicine, Department of Pharmacology, PO Box 245050, Tucson AZ 85724-5050, USA
ARTICLE INFO ABSTRACT
Article history:
Accepted 4 May 2009
Available online 8 May 2009
The rostroventromedial medulla (RVM) is an important source of descending modulatory
systems that both inhibit and facilitate pain at the level of the spinal cord. Noxious stimuli
can activate serotonergic neurons in the RVM and accelerate the turnover of 5-HT in the
spinal cord. While numerous studies suggest a bidirectional role for serotonergic
transmission at the spinal level, the subtypes of the 5-HT receptors that are associated
with descending facilitation or inhibition have not been clearly determined. Here, we
explore the relative contribution of spinal 5-HT7 and 5-HT3 receptors to antinociception or
hyperalgesia associated with states of enhanced net descending inhibition or facilitation
from the RVM. In uninjured rats, RVM microinjection of morphine produced dose-
dependent antinociception in the noxious thermal paw flick test while RVM
microinjection of CCK produced thermal hyperalgesia and tactile allodynia. Spinal
administration of the 5-HT7 antagonist SB-269970, but not of the 5-HT3 antagonist
ondansetron, blocked the antinociceptiveeffectsofRVMmorphine.Incontrast,
hyperalgesia induced by RVM-CCK was blocked by spinal ondansetron, but not by SB-
269970. The antinociceptive effects of systemic morphine were also blocked by spinal SB-
269970 but not ondansetron while hyperalgesia and allodynia resulting from SNL injury
were blocked by spinal ondansetron, but not SB-269970. These studies suggest that
descending pain inhibitory or facilitatory pathways from RVM act ultimately in the spinal
cord in acute and chronic pain states through activation of 5-HT7 and 5-HT3 receptors,
respectively.
© 2009 Elsevier B.V. All rights reserved.
Keywords:
Serotonergic
Pain
Descending facilitation
Inhibition
5-HT7
5-HT3
1. Introduction
The RVM is well recognized as a critical relay site for
integrating descending modulatory influences to the spinal
cord (Fields et al., 1999; Porreca et al., 2002; Suzuki et al., 2002).
The role of the RVM in the modulation of nociceptive inputs
has been extensively studied and this region is characterized
as an important source of descending modulatory systems
that both inhibit and facilitate pain at the level of the spinal
cord (Fields et al., 1999; Porreca et al., 2002; Suzuki et al., 2002).
Gaining an understanding of the spinal mechanisms of pain
inhibition or facilitation will allow greater insights into the
development of strategies for pain control. Opioid-induced
antinociception is mediated, in part, by spinally projecting
cells that terminate in the dorsal horn of the spinal cord,
producing inhibition of transmission of nociceptive
BRAIN RESEARCH 1280 (2009) 5259
Corresponding author.
E-mail address: michaelo@u.arizona.edu (M.H. Ossipov).
0006-8993/$ see front matter © 2009 Elsevier B.V. All rights reserved.
doi:10.1016/j.brainres.2009.05.001
available at www.sciencedirect.com
www.elsevier.com/locate/brainres
information (Fields et al., 1999; Millan, 2002). Conversely, it has
been hypothesized that activation of descending facilitation
under pathological conditions, such as peripheral nerve
injury, may provide a mechanism that maintains chronic
abnormal pain (Porreca et al., 2002; Vera-Portocarrero et al.,
2006). Both acute and chronic exposures to noxious stimuli
have been demonstrated to activate serotonergic neurons in
the RVM and to accelerate the turnover of 5-HT in the spinal
cord (Braz and Basbaum, 2008; Zhang et al., 2000). Although
numerous studies suggest a bidirectional role for serotonergic
transmission at the spinal level, the subtypes of the 5-HT
receptors that are associated with serotonergic modulation of
pain in the spinal dorsal horn have not been clearly
determined (Millan, 2002).
Currently, seven families of 5-HT receptors (5HT17),
including several subtypes within many of these families,
have been identified with selective excitatory and inhibitory
functions in neurons (Millan, 2002). Thus, there is a clear
implication that subtypes of the 5-HT receptor in spinal cord
could ultimately and selectively mediate the inhibitory and
facilitatory functions of descending pathways from the RVM.
However, because the spinal distribution of these receptor
subtypes have not been firmly established, and since highly
selective ligands for each of these receptors have not yet been
developed, our understanding of the role of these receptor
subtypes in spinal nociceptive transmission is still emerging.
The current understanding appears to be that the 5-HT1A, 5-
HT4 and 5-HT6 receptors do not modulate nociception at the
spinal level whereas spinal 5-HT1B and 5-HT2A appear to have
limited antinociceptive activity (see Bardin et al. 2000). Other
studies suggested that antinociception attributes to the 5-
HT1A receptor are in fact due to the 5-HT7 receptor (Brenchat
et al., 2009). Moreover, studies employing formalin-induced
flinch suggest that spinal 5-HT2A/C receptors are pronocicep-
tive (Kjorsvik et al., 2001), whereas studies employing
mechanical nociception suggest that these receptors may be
antinociceptive (Bardin et al., 2000). A similar contradiction
exists with regard to the 5-HT7 receptor. In one study, it was
shown that injection of the 5-HT antagonist into the hindpaw
attenuated the second (tonic), but not the first (acute), phase of
formalin-induced flinch (Rocha-Gonzalez et al., 2005). In
contrast, spinal administration of SB-269970 did not alter
either phase of formalin-induced flinch in the same study
(Rocha-Gonzalez et al. 2005). More recently, activation of the 5-
HT7 receptor produced dose-dependent antinociception in
mice (Brenchat et al., 2009) or rats (Harte et al., 2005), and was
implicated in the antinociceptive effect of morphine (Dogrul
and Seyrek, 2006). Differential results may be due to activity at
more than one receptor subtype by the agonists or antagonists
employed, as well as the nociceptive assays utilized.
It has been demonstrated that facilitatory projections from
the RVM enhance the responses of dorsal horn second order
neurons as well as the behavioral responses to noxious stimuli
through interaction with spinal 5-HT3 receptors (Suzuki et al.,
2002). Whether a spinal serotonergic receptor also plays a role
in the ultimate mediation of descending inhibition is not
known. Recently, the 5-HT7 receptor has been identified in the
CNS (Varnas et al., 2004). The function of central 5-HT7
receptors has not been clearly established, but based on the
localization pattern and functional studies using 5-HT7
receptor-selective antagonists, this receptor has been impli-
cated both in the regulation of circadian rhythms and in the
mechanisms underlying a number of psychiatric disorders
including anxiety and unipolar depression (Thomas and
Hagan, 2004; Varnas et al., 2004). Immunocytochemical
studies found that 5-HT7 receptors are localized in the
superficial layers of spinal cord dorsal horn which is consis-
tent with a predominant role of the 5-HT7 receptors in the
control of nociception (Meuser et al., 2002). Surprisingly, there
have been few attempts to directly examine the role of spinal
populations of 5-HT7 receptor in nociceptive processing. One
recent study demonstrated that morphine-induced antinoci-
ception is mediated in part by spinal 5-HT7, and not 5-HT1A or
5-HT2 receptors (Dogrul and Seyrek, 2006).
Because the RVM is a particularly important relay site for
integrating descending influences to the spinal cord, the
present study was undertaken to examine the contribution
of spinal 5-HT3 and 5-HT7 receptors in pain states which
predominately engage descending inhibition or facilitation
from this site. We used the selective 5-HT7 and 5-HT3 receptor
antagonist, SB-269970 (Hagan et al., 2000) and ondansetron
(Freeman et al., 1992) to dissect the role of spinal serotonergic
receptors in the expression of descending modulation. Here,
we provide evidence supporting the view that spinal 5-HT3
receptors ultimately mediate pain descending facilitation and
additionally demonstrate the critical importance of spinal 5-
HT7 receptors in the expression of descending inhibition.
2. Results
Subcutaneous morphine administration (1, 5 and 10 mg/kg, s.c.)
produced a dose-dependent antinociceptive effect in the paw
flick test (Fig. 1A). While i.th. SB-269970 (10 μg) given alone did not
produce any change in paw flick basal latencies, i.th. SB-269970
injected 30 min after the morphine injection produced a signi-
ficant, time-dependent reduction in the effect of morphine on
paw flick latencies (Fig. 1B). The effect of SB-269970 began within
15 min and peaked at 30 min post administration. Inhibition of
the antinociceptive effect of morphine by spinal SB-269970 lasted
90 min (Fig. 1B). i.th. ondansetron (10 μg) given alone did not
produce any change in paw flick basal latencies. Spinal admin-
istration of ondansetron (10 μg) did not attenuate the antinoci-
ceptive effect of morphine at any of the time-points tested
(Fig. 1C). The morphine doseresponse curves for data generated
at 30 min after the i.th. administration of SB-269970 and ondan-
setron (i.e.; 60 min after morphine) are illustrated in Fig. 1D.
Similar observations were made after microinjection of
morphine into the RVM. Morphine produced dose-dependent
antinociception which was evident at 30 min after microinjec-
tion (Fig. 2A). Spinally administered SB-269970 (10 μg) sharply
diminished the antinociceptive time-course of RVM morphine
(Fig. 2B). In contrast, spinal administration of ondansetron did
not alter the antinociceptive effect of RVM morphine at any of
the time-points tested (Fig. 2C). The morphine doseresponse
curves generated from the data at 30 min after the i.th.
administration of SB-269970 and ondansetron (i.e.; 60 min after
morphine) are illustrated in Fig. 2D.
The administration of CCK (50 ng) bilaterally into RVM
produced tactile allodynia and thermal hyperalgesia. Tactile
53BRAIN RESEARCH 1280 (2009) 5259
allodynia and thermal hyperalgesia were evident within10 min
of the microinjection of CCK as indicated by significant
reductions in paw withdrawal thresholds to probing with von
Frey filaments and paw withdrawal latencies to the noxious
thermal stimulus (Figs. 3A, B). Paw withdrawal thresholds were
significantly reduced to 3.28± 0.27 g from the mean baseline
value of 15±0 g and the mean paw withdrawal latencies to
infrared radiant heat were significantly reduced to 13.28± 1.5 s
from a mean baseline value of 17.4 ±1.1 s. The tactile allodynic
and thermal hyperalgesic effects of CCK (50 ng) lasted 60 min
and returned to baseline values at 90 min, where paw
withdrawal threshold and paw withdrawal latencies were
14.15±0.43 g and 17.28± 0.96 s, respectively. The spinal
administration of SB-269970 (10 μg, i.th.) alone did not produce
any change in mean paw withdrawal thresholds to tactile
stimuli or withdrawal latencies to noxious thermal stimuli
(Figs. 3A, B). Pretreatment with i.th. SB-269970 did not
attenuate the development of tactile allodynia (Fig. 3B) and
thermal hyperalgesia (Fig. 3B) induced by microinjection of
CCK into the RVM. There were no significant differences
observed between the groups receiving spinal saline or SB-
269970. In contrast, spinal administration of ondansetron
(10 μg, i.th.) completely abolished both tactile allodynia and
thermal hyperalgesia (Figs. 3A, B) in RVM-CCK injected
animals, whereas ondansetron (10 μg, i.th.) alone did not elicit
any change in baseline paw withdrawal threshold and latency
in control animals.
Tactile allodynia and thermal hyperalgesia were estab-
lished 7 days after SNL. Mean paw withdrawal thresholds of
rats with SNL was significantly reduced to 1.6 ± 0.7 g from a
pre-SNL baseline value of 15±0 g and mean paw withdrawal
latencies were significantly reduced to 12.3± 0.8 s from a mean
pre-SNL baseline value of 16.5 ± 1.2 s (Figs. 4A, B). The spinal
administration of SB-269970 (10 μg, i.th.) alone did not produce
any change in baseline paw withdrawal threshold and latency
in sham-operated animals. Furthermore, spinal administra-
tion of SB-269970 pretreatment did not affect tactile allodynia
and thermal hyperalgesia in rats with SNL. In contrast, spinal
administration of ondansetron (10 μg, i.th.) completely abol-
ished behavioral signs of tactile allodynia and thermal
hyperalgesia (Figs. 4A, B). Paw withdrawal thresholds to tactile
stimuli and paw withdrawal latencies to noxious thermal
stimuli were significantly elevated when compared to those of
rats with SNL without ondansetron, and were similar in
magnitude to the behavioral responses observed in sham-
operated rats (Fig. 4). Finally, spinal administration of
Fig. 1 The antinociceptive effects of 1, 5 and 10 mg/kg of s.c. morphine are shown (A), along with the effects of i.th.
administration of SB-269970, a 5-HT7 receptor antagonist (B) and ondansetron, a 5-HT3 receptor antagonist (C) on the
antinociceptive responses induced by s.c. morphine in the paw flick test. Baseline paw flick latencies were determined in male
SpragueDawley rats that then received morphine (1, 5 and 10 mg/kg, s.c.). SB-269970, ondansetron (10 μg/10 μl) or saline were
given i.th. 30 min following s.c. morphine administration. N=68 for each group. * differences corresponding to dose of
morphine alone p<0.05. Paw flick latencies at the time of peak blocking effects of SB-269970 and ondansetron (30 min) given
i.th. 30 min following s.c. morphine (1, 5 and 10 mg/kg, s.c.) administration were used in order to generate the doseresponse
curve (D). N=68 for each group.
54 BRAIN RESEARCH 1280 (2009) 5259
ondansetron did not alter behavioral responses to light tactile
stimuli or noxious thermal stimuli in sham-operated rats.
3. Discussion
The results of present study demonstrate that the antinocicep-
tive effects of morphine administered eithersystemically or into
the RVM were blocked by intrathecal administration of an
antagonist at the 5-HT7, but not the 5-HT3, receptor. These
results indicate that activation of spinal 5-HT7 receptors is
ultimately critical in the expression of opiate-induced anti-
nociception, likely through activation of descending inhibition.
In contrast, tactile allodynia and thermal hyperalgesia induced
either pharmacologically by microinjection of CCK into the RVM
or by SNL were abolished by spinal administration of the 5-HT3,
but not by the 5-HT7, receptor antagonist. These findings are
consistent with ultimate blockade of descendingpronociceptive
influences arising from the RVM, as suggested by Dickenson et
al. (Bee and Dickenson, 2007; Suzuki et al., 2002).
The antinociceptive effects of morphine are thought to arise
from activation of opiate receptors present on neurons in brain,
spinal cord dorsal horn, and on peripheral terminals of primary
afferents (Taylor and Basbaum, 2003). Critically, a powerful
synergistic interaction between spinal and supraspinal mor-
phine has been demonstrated, underscoring the role of activity at
multiple sites within the CNS required for the full expression of
morphine-induced antinociception (Yeung and Rudy, 1980).
Spinal transection markedly attenuated, but did not abolish,
the antinociceptive effect of systemic morphine, indicating that
morphine exerts its antinociceptive effect through spinal
mechanisms as well as through the activation of descending
pain inhibitory systems (Advokat and Burton, 1987). Further
studies have established that the antinociceptive effects of
systemically administered morphine are mediated in part by
descending bulbospinal projections that inhibit dorsal horn
neuronal responses to noxious stimuli (Clarke and Ward, 2000;
Lo et al., 2004; Xu et al., 1994). While the importance of activation
of descending inhibition is recognized as a major part of
morphine antinociception, the precise consequences of such
descending influence has not been identified. The antinocicep-
tive actions of opiates have been linked to spinal 5-HT1a, and
subtypes of the 5-HT2, but not 5-HT3 receptors (Clarke and Ward,
2000; Liu et al., 2002; Lo et al., 2004; Xu et al., 1994). In our study, we
Fig. 2 The antinociceptive effects of morphine (1, 5 and 10 μg) microinjected into the RVM (A) are shown along with the effects
of i.th. administration of SB-269970, a 5-HT7 receptor antagonist (B) and ondansetron, a 5-HT3 receptor antagonist (C) on the
antinociceptive responses induced by intra-RVM morphine in the paw flick test. Baseline paw flick latencies were determined
in male SpragueDawley rats that then received intra-RVM morphine (1, 5 and 10 μg/rat). SB-269970 (10 μg/10 μl) or saline were
given i.th. 30 min following the morphine administration. (*) Denotes differences corresponding dose of morphine alone
p<0.05. N=68 for each group. Paw flick latencies at the time of peak blocking effects of SB-269970 and ondansetron (30 min)
given i.th. 30 min following morphine microinjection into the RVM (1, 5 and 10 μg/rat) were used in order to generate the
doseresponse curve (C). N=68 for each group.
55BRAIN RESEARCH 1280 (2009) 5259
also found that spinal 5-HT3 receptors do not appear to play an
important role in the antinociceptive effect of systemic mor-
phine. However, we found that the spinal administration of the
selective 5-HT7 receptor antagonist SB-269970 markedly attenu-
ated that antinociceptive effect of systemic morphine, indicating
a novel role of the 5-HT7 receptor in mediating morphine-
induced antinociception. These results are consistent with our
previous findings in the mouse (Dogrul and Seyrek, 2006).
The rostral ventromedial medulla (RVM) is generally con-
sidered to be the final common relay through which descending
inhibitory and facilitatory bulbospinal projections may attenu-
ate or enhance spinal nociceptive transmission (Bee and
Dickenson, 2007). Notably, the analgesic actions of systemic
morphine are in large part mediated by activation of brainstem
neurons that exert a net inhibitory effect on nociceptive
processing in the spinal dorsal horn (Bee and Dickenson, 2007;
Heinricher et al., 2001). The dose-dependent antinociception
produced by microinjection of morphine into the RVM observed
in the present investigation is consistent with the activation of
descending inhibitory fibers from the RVM. Importantly, it was
also observed that the antinociception elicited by morphine
given into the RVM was markedly attenuated by the selective 5-
HT7 antagonist, SB-269970, but not by the 5-HT3 antagonist
ondansetron. Thus, the results of the present investigation
suggest that morphine given systemically or into the RVM
activates descending inhibitory projections that ultimately
release 5-HT in the spinal cord, where serotonin may act at
cells expressing 5-HT7 receptors to inhibit transmission of
nociceptive signals in the spinal dorsal horn.
The biologic role of spinal 5-HT receptors is based on
results from studies on the anatomical distribution of 5-HT
receptors (Millan, 2002). Meuser et al. (2002) demonstrated that
Fig. 3 The effects of i.th. SB-269970, a 5-HT7 receptor
antagonist and ondansetron, a 5-HT3 receptor antagonist
intra-RVM-CCK induced tactile allodynia (A) and thermal
hyperalgesia (B). Baseline paw withdrawal threshold to
probing with von Frey filaments (A) and latencies to thermal
stimulus (B) were determined in male SpragueDawley
rats prior to and at select time-points after CCK (50 ng)
microinjection into RVM. SB-269970 and ondansetron (10 μg/
10 μl) were given i.th. 5 min before CCK administration. (*)
denotes differences from RVM saline+ i.th. saline. (+) denotes
differences from RVM-CCK+i.th. saline p< 0.05. N=68 for
each group.
Fig. 4 The effects of i.th. SB-269970, a 5-HT7 receptor
antagonist and ondansetron, a 5-HT3 receptor antagonist on
tactile allodynia (A) and thermal hyperalgesia (B) induced by
spinal nerve ligation. Male SpragueDawley rats received L5/
L6 spinal nerve ligation. Paw withdrawal thresholds to
probing with von Frey filaments (A) and latencies to thermal
stimulus (B) were determined prior to and at select time-
points after the i.th. injection of SB-269970 and ondansetron
(10 μg/10 μl). (*) differences from sham+ i.t.h saline. (+)
differences from SNL+i.t.h saline p< 0.05. N=68 for each
group.
56 BRAIN RESEARCH 1280 (2009) 5259
5-HT7 receptors are localized in the superficial layers of spinal
cord dorsal horn whereas a second study indicated that 5-HT7
receptors appear to be predominant in the intermediate
regions of the spinal cord. Doly et al. (2005) showed the
cellular and subcellular distribution of the 5-HT7 receptor in
the spinal cord and found that 5-HT7 receptors were mainly
localized on primary afferent fibers, peptidergic interneurons
in laminae I and II, and on glial cells. Thus, neuroanatomical
localization of 5-HT7 receptors in the spinal cord suggests the
importance of 5-HT7 receptors in pain inhibitory pathways,
consistent with our study.
Work by Liu et al. (2002) showed that the spinal adminis-
tration of 5-HT
1A
receptor antagonist blocked the antinoci-
ceptive effects of supraspinal morphine. Although the
pharmacology of the 5-HT7 receptor is distinct and does not
correspond to any previously characterized subtype, it does
exhibit high affinity for a number of ligands that interact with
the 5-HT1A receptor family. Thus, great caution must be taken
when trying to ascribe results to one particular receptor
subtype over another (Meuser et al., 2002). For instance, the 5-
HT7 receptor exhibits relatively high affinity for the classic 5-
HT1A receptor agonist, 8-OH-DPAT, suggesting that multiple
functions formerly attributed to a 5-HT
1A
-like receptor may be
likely attributable to the 5-HT7 subtype (Bonaventure et al.,
2002). Additionally, some studies demonstrated the absence of
5-HT1A receptor mRNA in rat DRG (Chen et al., 1998), while
detecting the 5-HT7 receptor subtype in both rat (Pierce et al.,
1996) and human DRG (Freeman et al., 1992). In this study we
used the potent and selective 5-HT7 receptor antagonist SB-
269970 displayed at least 100-fold selectivity over 5-HT
1A
receptor (Hagan et al., 2000; Lovell et al., 2000). It is therefore
expected that the doses of SB-269970 employed in the present
investigation selectively blocked spinal 5-HT7 receptors. The
5-HT7 receptors have been shown to stimulate cAMP forma-
tion and to elicit neuronal excitation (Cardenas et al., 1999).
Thus, it is reasonable to suggest that systemic morphine
activates supraspinal neurons projecting to the spinal cord
resulting ultimately in the release of 5-HT. However, the
mechanism by which activation of 5-HT7 receptors results in
inhibition is not known and will require further study.
Damage to peripheral nerves is thought to result in the
development of peripheral and central sensitization which
can manifest behaviorally as thermal hyperalgesia and tactile
allodynia. It has been repeatedly demonstrated that the
activation of supraspinal facilitatory pathways from the RVM
maintains the abnormal, enhanced pain state associated with
peripheral nerve injury (Bee and Dickenson, 2007; Millan, 2002;
Porreca et al., 2002; Zhang et al., 2000). Moreover, it has been
shown that increased availability of CCK in the RVM second-
ary to SNL may drive descending facilitation, which manifests
behaviorally as tactile allodynia or thermal hyperalgesia. For
example, microinjection of the CCK2 receptor antagonist into
the RVM abolished behavioral signs of neuropathic pain in rats
with SNL (Kovelowski et al., 2000). Conversely, microinjection
of CCK into the RVM of naive rats elicited tactile hyperesthesia
and thermal hyperalgesia (Kovelowski et al., 2000; Xie et al.,
2005) and such hyperalgesia was blocked by lesions of the
dorsolateral funiculus (Xie et al., 2005), consistent with the
results of the present investigation. Interestingly, we demon-
strated that spinal administration of ondansetron markedly
attenuated tactile allodynia and thermal hyperalgesia induced
both after SNL or microinjection of CCK into the RVM. Recent
electrophysiological studies demonstrated that peripheral
nerve injury is associated with an increased descending
facilitation that enhances the responses of spinal dorsal
horn neurons, and that this enhancement is mediated
through the activation of spinal 5-HT3 receptors (Kovelowski
et al., 2000; Suzuki et al., 2002). The present finding of a
selective attenuation of tactile allodynia and thermal hyper-
algesia in rats with SNL or with microinjection of CCK into the
RVM by spinal ondansetron provides further evidence sup-
porting the hypothesis that descending facilitation from the
RVM includes a serotonergic component that is mediated
through activation of spinal 5-HT3 receptors. We note that
although only a single dose of ondansetron was employed,
this dose blocked behavioral manifestations of descending
facilitation and thus was likely to sufficiently occupy the 5-
HT3 receptors. Blockade of 5-HT3 alone was insufficient to
affect the antinociceptive effect of morphine.
These experiments provide pharmacological evidence that
descending pain modulatory pathways arising from the RVM
exert bidirectional influence upon nociception ultimately
through activation of different serotonergic receptors in the
spinal cord. It appears that descending inhibition mediates
antinociception through activation of spinal 5-HT7 receptors
whereas descending facilitation mediates enhanced pain
through activation of spinal 5-HT3 receptors. Such information
may help in the design of novel therapeutic strategies for pain.
4. Experimental procedures
4.1. Animals
Adult, male SpragueDawley rats (250300 g) were used. They
were placed in a quiet, humidity controlled room (22 ±3 °C and
60±5%, respectively) in which a 12/12 h lightdark cycle was
maintained (lights on from 8 am to 8 pm). All of the testing was
performed in accordance with policies and recommendation
of IASP and NIH guidelines for the handling and use of
laboratory animals.
All drugs were freshly prepared by dissolving them in
saline. Drug solutions were prepared so that the desired dose,
expressed in terms of salt, was contained in a volume 2 ml/kg
of body weight for subcutaneous (s.c.) injection, in 10 μl for
intrathecal (i.th.) injection and 0.5 μl for intra-RVM injection.
4.2. Surgical procedures
While under halothane surgery, rats were implanted with
intrathecal catheters (PE-10, 7.5 cm) as described by Yaksh and
Rudy (1976) for the administration of serotonin receptor
antagonists at the level of the lumbar spinal cord. Animals
were allowed to recover for 4 days. Following i.th. surgery,
groups of animals were also implanted with guide cannula for
microinjection into the RVM or were subjected to SNL.
4.2.1. RVM surgery
Rats were prepared for bilateral RVM drug administration by
placing anesthetized (80 mg/kg of ketamine and 12 mg/kg of
57BRAIN RESEARCH 1280 (2009) 5259
xylazine, i.p) animals in a stereotaxic headholder. The skull
was exposed and two 26-gauge guide cannula separated by
1.2 mm (Plastics One Inc., Roanoke, VA) were directed toward
the lateral portions of the RVM (AP11.00 mm from bregma, L
±0.6 mm; DV8.5 mm from the base of the skull (Paxinos and
Watson, 1986). The guide cannula were cemented in place and
secured to the skull by small stainless steel machine screws.
The animals were allowed to recover for 5 days before any
drug injection. At the termination of the experiments,
pontamine blue was injected into the site of the RVM
injections, and cannula placement was verified histologically.
4.3. Nerve injury model
Spinal nerve injury was performed according to the procedure
of Kim and Chung (1992). Anesthesia was induced with 2%
halothane in O
2
at 2 l/min and maintained with 0.5% halothane
in O
2
. After surgical preparation of the rats and exposure of the
dorsal vertebral column from L4 to S2, the L5 and L6 spinal
nerves were tightly ligated distal to the dorsal root ganglion
using 4-0 silk suture. The incision was closed and the animals
were allowed to recover for five days. Sham surgery was
performed by exposing the spinal nerves without ligation.
4.4. Thermal nociceptive tests
The paw flick test was used to assess nociception. Rats were
allowed to acclimate to plexiglass chambers placed on a glass
plate. An infrared (IR) beam was focused onto the plantar
aspect of the hindpaw at an intensity that produced mean
baseline paw flick latencies ranging from 3.7 to 4.5 s (Ugo
Basile, Italy). A cut-off time of 10 s was used to prevent tissue
damage. Doseresponse curves were constructed from the
time of peak effect determined after s.c. or intra-RVM admin-
istration of morphine. For studies designed to explore thermal
hyperalgesia, the IR intensity was adjusted to produce mean
baseline paw withdrawal latencies ranging between 15 and
18 s. The withdrawal latencies were determined both before
and after RVM-CCK administration or SNL.
4.5. Drug administration
Morphine or CCK was administered bilaterally (injection
volume of 0.5 μl per side) into the RVM through a previously
implanted guide cannula to otherwise uninjured rats. RVM
injections were performed by slowly expelling of drug solution
through a 33 gauge injection cannula inserted through the
guide cannula and protruding an additional 1 mm into fresh
brain tissue to prevent back flow of drug into the guide
cannula. Saline was injected into the RVM as a control. In the
antinociception studies, SB-269970 and ondansetron were
given i.th. 30 min after systemic or intra-RVM morphine
administration. In those studies where CCK was microinjected
into the RVM, SB-269970 and ondansetron were given i.th.
10 min before the intra-RVM injection.
4.6. Tactile allodynia
The paw withdrawal thresholds of the hindpaws of the rats
were determined in response to probing with eight calibrated
von Frey filaments (Stoelting, Wood Dale, IL) in logarithmically
spaced increments ranging from 0.41 to 15 g (4150 mN). Each
filament was applied perpendicularly to the plantar aspect of
the hindpaw ipsilateral to SNL or to the left hindpaws of RVM-
CCK administered rats. Withdrawal thresholds were deter-
mined by sequentially increasing and decreasing the stimulus
strength (up and downmethod), analyzed using a Dixon
non-parametric test (Chaplan et al., 1994) and expressed as the
mean paw withdrawal threshold.
4.7. Drugs
Ondansetron (Zofran, Western Medical Supply, Inc, Anahein
CA), a highly selective 5-HT3 receptor antagonist with a
selectivity ratio of approximately 1000:1 compared with
affinities for other receptors (Freeman et al., 1992) and SB-
269970 (Sigma Chemical Co., St. Louis, MO), a selective 5-HT7
antagonist with displaying over 100-fold selectivity versus
other 5-HT receptors (Lovell et al., 2000) were used.
4.8. Statistical analysis
The data were expressed as the mean ± S.E.M. in all cases.
Groups of 68 rats were tested at each dose. A non-parametric
method of statistical analysis was used. Statistical signifi-
cance of more than two groups were evaluated by Kruskall
Wallis test (p<0.05), followed by Dunnett's multiple test for
individual comparisons.
Acknowledgments
Dr. Ahmet Dogrul thanks the Turkish Brain Research Society
for a travel award.
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59BRAIN RESEARCH 1280 (2009) 5259
    • "Descending pain facilitation. The descending pain modulation from the PAG and rostral ventromedial medulla has clearly been involved in the facilitation of nociceptive signaling in different models of persistent pain (Donovan-Rodriguez et al., 2006; Svensson et al., 2006; Dogrul et al., 2009) as well as in the development of hyperalgesia and analgesic tolerance induced by opiates (Vanderah et al., 2001). The implication of descending pathways originating from the rostral ventromedial medulla (RVM) was investigated further in naı¨venaı¨ve and paw incision-sensitized rats. "
    [Show abstract] [Hide abstract] ABSTRACT: Opioids produce strong analgesia but their use is limited by a paradoxical hypersensitivity named opioid-induced hyperalgesia (OIH) that may be associated to analgesic tolerance. In the last decades, a significant number of preclinical studies have investigated the factors that modulate OIH development as well as the cellular and molecular mechanisms underlying OIH. Several factors have been shown to influence OIH including the genetic background and sex differences of experimental animals as well as the opioid regimen. Mu opioid receptor (MOR) variants and interactions of MOR with different proteins were shown important. Furthermore, at the cellular level, both neurons and glia play a major role in OIH development. Several neuronal processes contribute to OIH, like activation of neuroexcitatory mechanisms, long-term potentiation (LTP) and descending pain facilitation. Increased nociception is also mediated by neuroinflammation induced by the activation of microglia and astrocytes. Neurons and glial cells exert synergistic effects, which contribute to OIH. The molecular actors identified include the Toll-like receptor 4 and the anti-opioid systems as well as some other excitatory molecules, receptors, channels, chemokines, pro-inflammatory cytokines or lipids. This review summarizes the intracellular and intercellular pathways involved in OIH and highlights some mechanisms that may be challenged to limit OIH in the future.
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    • "Pharmacological and genetic tools targeting the 5-HT 7 receptor in animal models have implicated this receptor in various pathophysiological processes, including: regulation of body temperature [20], circadian rhythms [21], learning and memory [22] . Inflammatory processes in the CNS [23], smooth muscle relaxation of cerebral arteries [24], mood disorders [25, 26], and pain [27]. The Na + -K + pump is a ubiquitous membrane protein that catalyzes the active transport of K + into cells and Na + out of cells against their electrochemical gradient. "
    [Show abstract] [Hide abstract] ABSTRACT: The observed controversy that N-(4-cyanophenylmethyl)-4-(2-diphenyl)-1-piperazinehexanamide (LP-211), a selective serotonin (5-HT 7 ) receptor agonist, may either modify or exacerbate imbalances in serum electrolyte concentrations and renal tissue of spinal cord trauma cases has not been reported yet. The aim of this study was to better understand the effects of a new 5-HT 7 receptor agonist, LP-211, on serum electrolyte changes in spinal cord injured- (SCI) rats. Methods Sixty male rats were assigned to the following groups: A) Intact (saline as vehicle, 1 ml/kg, i.p.), B) Intact [LP-211, (0.003–0.3 mg/kg, i.p.)], C) Sham-operated [laminectomy + vehicle (1 ml/kg, i.p.)], D) Sham-operated [laminectomy + LP-211 (0.003–0.3 mg/kg, i.p.)], E) Treatment [laminectomy + spinal trauma (SCI) + vehicle (1 ml/kg, i.p.)], F) Treatment [laminectomy + spinal trauma + LP-211 (0.003–0.3 mg/kg, i.p.)]. SCI was performed by placing an aneurysm clip, extradurally at the level of T10. After two weeks, LP-211 was administered cumulatively and each dose was injected (i.p.) with 20 min interval. At the end of the experiment, blood samples were collected for biochemical evaluations of the electrolytes employing standard commercial kits. Results The present results indicate elevated serum levels of Na + , K + , and Mg 2+ in SCI rats and significant differences demonstrated between the groups [P < 0.001, F(5, 35) = 23.92], [P < 0.001, F(5, 35) = 67.63], [P < 0.001, F(5, 35) = 71.144], respectively. So that, in groups B, D and F, there was a significant increase in K + and Mg 2+ serum levels compared to the groups A, C, and E (P < 0.001). Furthermore, Na + serum levels in SCI (LP-211), laminectomy (LP-211), and intact (LP-211) groups tended to be statistically lower than SCI (saline), laminectomy (saline) and intact (saline) groups. Infact, hyponatremia, hyperkalemia and hypermagnesemia was obtained in group F. Nevertheless, in the remaining measured serum electrolytes such as calcium (Ca 2+ ), iron (Fe 2+ ) and phosphorus (P 3− ), chlorine (Cl − ), copper (Cu + ), and zinc (Zu + ), no significant changes were observed. Conclusion It was shown that acute additive LP-211 treatments in the SCI group led to hyponatremia, hyperkalemia and hypermagnesemia, it may be stated that LP-211 treatment as a promising candidate for treating SCI complications in some systems especially urinary tract might take into consideration and further studies would be needed to clarify its benefits or drawbacks. The observed discrepancies, nevertheless; will also pose new questions. Altogether, this will ultimately contribute to further understanding the pathophysiological role regarding 5-HT 7 receptor activation.
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    • "Further, inflammatory cytokines could directly modulate 5-HT activity by enhancing synthesis, altering trafficking of receptors or transmitters, or changing channel conductance. Numerous studies suggest an inhibitory role for serotonergic transmission from RVM to spinal cord dorsal horn, and the subtypes of the 5-HT receptors associated with this modulation are investigated mainly at spinal level [17,54]. However, serotonergic fibers and terminals are found throughout RVM [71], as are a number of 5-HT receptor subtypes [39,57,58,77]. "
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