1Department of Anesthesiology, 2Department of Pharmacology, and the Graduate Program in Neurobiology and Behavior, University of Washington School of Medicine, Seattle, Washington 98195-6540
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ABSTRACT |
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Terman, Gregory W., Clifford L. Eastman, and Charles Chavkin. Mu Opiates Inhibit Long-Term Potentiation Induction in the Spinal Cord Slice. J. Neurophysiol. 85: 485-494, 2001. Long-term potentiation (LTP) involves a prolonged increase in neuronal excitability following repeated afferent input. This phenomenon has been extensively studied in the hippocampus as a model of learning and memory. Similar long-term increases in neuronal responses have been reported in the dorsal horn of the spinal cord following intense primary afferent stimulation. In these studies, we utilized the spinal cord slice preparation to examine effects of the potently antinociceptive mu opioids in modulating primary afferent/dorsal horn neurotransmission as well as LTP of such transmission. Transverse slices were made from the lumbar spinal cord of 10- to 17-day-old rats, placed in a recording chamber, and perfused with artificial cerebrospinal fluid also containing bicuculline (10 µM) and strychnine (1 µM). Primary afferent activation was achieved in the spinal slice by electrical stimulation of the dorsal root (DR) or the tract of Lissauer (LT) which is known to contain a high percentage of small diameter fibers likely to transmit nociception. Consistent with this anatomy, response latencies of LT-evoked field potentials in the dorsal horn were considerably slower than the response latencies of DR-evoked potentials. Only LT-evoked field potentials were found to be reliably inhibited by the mu opioid receptor agonist [D-Ala2, N-Me-Phe4, Gly5] enkephalin-ol (DAMGO, 1 µM), although evoked potentials from both DR and LT were blocked by the AMPA/kainate glutamate receptor antagonist 6-cyano-7-nitroquinoxalene-2,3-dione. Moreover repeated stimulation of LT produced LTP of LT- but not DR-evoked potentials. In contrast, repeated stimulation of DR showed no reliable LTP. LTP of LT-evoked potentials depended on N-methyl-D-aspartate (NMDA) receptor activity, in that it was attenuated by the NMDA antagonist APV. Moreover, such LTP was inhibited by DAMGO interfering with LTP induction mechanisms. Finally, in whole cell voltage-clamp studies of Lamina I neurons, DAMGO inhibited excitatory postsynaptic current (EPSC) response amplitudes from LT stimulation-evoked excitatory amino acid release but not from glutamate puffed onto the cell and increased paired-pulse facilitation of EPSCs evoked by LT stimulation. These studies suggest that mu opioids exert their inhibitory effects presynaptically, likely through the inhibition of glutamate release from primary afferent terminals, and thereby inhibit the induction of LTP in the spinal dorsal horn.
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INTRODUCTION |
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The CNS has been found capable
of remarkable plasticity, demonstrating functional and structural
changes as a result of incoming stimuli, which then alter the response
of that neural system to future stimuli. Such neuroplasticity may play
a role in such diverse phenomena as neural protection and regeneration
following stroke, epileptogenesis, opiate tolerance, learning and
memory, and certain chronic pain states (e.g., Dobkin
1993). The best-studied model of activity-dependent synaptic
plasticity, called long-term potentiation (LTP), involves a prolonged
increase in neuronal excitation following repeated afferent input to
that neuron and has been suggested to be a cellular basis for learning
(Bliss and Collingridge 1993
). LTP has been most
extensively studied in the hippocampus due to the role this region is
thought to play in learning and memory and to the highly structured
cytoarchitecture of the hippocampus that permits selective afferent
stimulation not only in vivo but also in vitro using slice
preparations. However, LTP-like phenomena have also been reported in
other regions of the neuraxis, including the dorsal horn of the spinal
cord in neural circuits likely to mediate nociception (e.g.,
Randic et al. 1993
; Woolf et al. 1989
).
For nearly 50 years (Hardy et al. 1952; Mendell
1966
) increased pain responses following tissue injury have
been hypothesized to occur in part due to changes that occur in the
spinal cord dorsal horn. These changes have collectively been termed
central sensitization and may last hours to days after cessation of or anesthetic blockade of a repeated nociceptive stimulus (e.g., Coderre and Katz 1997
; Woolf 1983
).
Central sensitization has now been demonstrated using a number of
response end points (e.g., stimulus threshold, response frequency,
receptor field size) and eliciting stimuli (e.g., noxious heat, noxious
chemicals, acute joint inflammation and C-fiber electrical stimulation)
[for review, see Coderre 1993 No. 5 (Baranauskas
and Nistri 1998
)]. Moreover, a number of studies have
indicated that this "central sensitization" in pain transmitting
systems may have clinical significance (e.g., Woolf and Doubell
1994
). Intraspinal opioids, for example (Katz et al.
1992
), given prior to surgical incision have been reported to
reduce postoperative pain and analgesic requirements more than the same
dose of drug given after incision
presumably after central sensitization has been induced.
Results of many studies of central sensitization show similarities to
studies of LTP in the hippocampus. For example,
N-methyl-D-aspartate (NMDA) receptor antagonists
have been reported to block both hippocampal LTP (Harris et al.
1984) and a number of electrophysiological (Liu and
Sandkuhler 1998
; Woolf and Thompson 1991
) and
behavioral (Coderre and Melzack 1992
; Mao et al.
1992
; Ren et al. 1992
; Seltzer et al.
1991
) models of central sensitization. However, since the mechanisms underlying LTP have been shown to differ dramatically from
region to region, even within the hippocampus (including NMDA
dependence) (Johnston et al. 1992
), detailed studies of
nociceptive transmission and neuroplasticity within the spinal cord
will likely be required to elucidate the mechanisms modulating spinal sensitization.
In the last few years, a number of in vitro studies of long-lasting
spinal dorsal horn synaptic plasticity have been published, investigating the inducing stimuli, modulatory pharmacology, and cellular or molecular mechanisms activated during sensitization (e.g.,
Garraway et al. 1997; Hori et al. 1996
;
Pockett 1995
; Randic et al. 1993
;
Terman and Chavkin 1997
). In this paper, we describe a
reliable model of LTP in the neonatal rat spinal cord slice preparation, characterize its dependence on both NMDA and non-NMDA glutamate receptors, and report its modulation by mu opioids acting on
LTP induction mechanisms, likely at presynaptic sites on primary afferents. Portions of this data have already been presented in abstract form (Terman and Chavkin 1997
).
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METHODS |
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Ten- to 17-day-old rats were anesthetized with halothane, and a laminectomy was performed from mid-thoracic to low lumbar levels. Cold buffer [(in mM) 220 sucrose, 3 KCL, 8 MgCl2, 1.25 NaH2PO4, 26 NaHCO3, and 10 D-glucose bubbled with 95% O2-5% CO2] was placed on the spinal cord, decapitation was performed, and the cord quickly removed and placed in 35°C, 2% low melting point agar. The spinal cord embedded in agar was then chilled, blocked, and sliced (300-400 µm) using a Vibratome (Ted Pella). Spinal cord slices with attached dorsal root remnants were transferred to an O2-bubbled, Plexiglas recording chamber where they were perfused with modified artificial cerebrospinal fluid (ACSF) [(in mM) 113 NaCl, 3 KCl, 1 NaH2PO4, 25 NaHCO3, 11 glucose, 1 CaCl2, and 2 MgCl2 plus bicuculline (10 µM) and strychnine (1 µM)] at a rate of 1 ml/min for 1 h prior to electrophysiological recordings.
With the aid of a dissecting microscope, a 3 M NaCl containing glass
recording electrode (approximately 5-10 M) was advanced into the
medial region of the base of the ipsilateral spinal dorsal horn (Fig.
1). Additionally, one suction electrode
(AM Systems) was attached to the dorsal root remnant and another was
placed in the tract of Lissauer (the white matter capping the
dorsal horn). This latter electrode was aimed at the tract of
Lissauer's most ventrolateral border to minimize current spread to the
dorsal root and the recording site.
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Quantitation of responses
A digitizing oscilloscope (Tektronix, Beaverton, OR) was used to measure stimulus-evoked peak-to-peak response amplitudes recorded using a Axopatch 200 amplifier (Axon), and the recording electrode was advanced to maximize these responses. The dorsal root (DR) and the Lissauer's tract (LT) electrodes were sequentially stimulated (Grass S88) once every minute with a 0.5-ms pulse at the current intensity which produced an approximately half-maximal peak to peak field potential amplitude (S1/2).
In LTP experiments, 12 1-s trains of 10-Hz, 0.5-ms pulses at the S1/2
intensity, given 1 train every 10 s were applied to the specified
site. LTP was operationally defined as the mean change in field
potential response amplitude for five S1/2 intensity stimuli given
beginning 30 min after tetanic stimulation compared with the mean
response to five test pulses given immediately prior to the 10-Hz
stimulation (Baseline). Thus % Potentiation = [(post-tetanus amplitude/baseline amplitude) 1]*100.
Drug effects were studied at equilibrium from 10 to 15 min after
addition of drug to the ACSF perfusate. The mean field potential amplitude of five S1/2 intensity stimuli given immediately prior to
drug application was compared with the mean response to five stimuli
given beginning 10 min postdrug. Some data were transformed to % Baseline Amplitude [(post-treatment mean amplitude/mean baseline amplitude)*100] and some to % Amplitude Change {[(postdrug predrug mean amplitude)/predrug mean amplitude]*100} for
illustrative purposes and statistically analyzed using between subjects
and/or within subjects analyses of variance as appropriate (Statistica software) with Tukey's tests for between group post hoc comparisons. A
probability of greater than 0.05 was chosen for statistically significant rejection of the null hypothesis.
Whole cell voltage-clamp studies
Slice preparation was identical to that detailed in the
preceding text, but slices were held in place in the experimental chamber with a harp constructed of platinum wire in the shape of a
"U" with nylon threads glued across the open end of the U at
intervals smaller than the spinal cord diameter. A Zeiss Axioskop FS
microscope with a 770 (±40)-nm long-pass filter was used to identify
specific cells from which to record. A glass recording electrode
[approximately 15 MX when filled with (in mM) 125 KMeSO4, 8 NaCl, 10 HEPES, 2 MgATP, 0.5 NaGTP, and
5 EGTA at pH 7.3 and approximately 285 mOsm] was advanced using a
micro-manipulator (Sutter), a CCD camera (Cohu) and a black and white
monitor (Sony) until whole cell configuration was obtained using
suction on the targeted cell. Whole cell voltage clamp was maintained
at a resting potential of 70 mV except as dictated by the particular
experiment using an Axopatch 200 amplifier (Axon Instruments) and
pclamp 6.0 software (Axon Instruments). Initial series resistance and capacitance were noted and a change of greater than 20% was used as
the upper limit for inclusion of data in analysis. Data analysis of
peak postsynaptic currents was performed using pclamp 6.0 software and
statistical software as in the preceding text.
In glutamate puff experiments, in addition to the recording pipette, a second pipette, containing 100 mM glutamate (Sigma), was placed near the voltage-clamped cell. Tract of Lissauer stimulation was alternated with pressure ejection (Picospritzer, General Valve, Fairfield, NJ; 20-40 psi, 50 ms) every 2 min and the evoked responses (EPSCs) recorded.
In paired-pulse experiments, 30 paired (60 ms interstimulus interval) LT electrical stimuli were delivered at 30-s intervals during baseline, [D-Ala2, N-Me-Phe4, Gly5] enkephalin-ol (DAMGO, 1 µm) perfusion, and DAMGO plus naloxone (1 µm) treatments. Paired evoked EPSC amplitudes were converted to a ratio [P2/P1 = (2nd peak response amplitude)/(1st peak response amplitude)] for each stimulus pair, and these were averaged for each treatment for each cell studied. A Friedman test was used for statistical analysis of these repeated measure ratios.
Materials
Bicuculline methiodide, strychnine,
6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and
D2-amino-5-phosphonovalerate (APV) were purchased from
Sigma Chemical. DAMGO was purchased from Research Biochemicals.
Naloxone was a gift from NIDA. All drugs were added to the perfusate in
a 1:1000 dilution.
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RESULTS |
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Previous studies by others have demonstrated LTP of field
potentials in the spinal dorsal horn in vivo (Liu and Sandkuhler 1995) and in vitro (Pockett 1995
). Our initial
studies were directed at pharmacologically characterizing the field
potentials elicited by stimulation of LT and DR in spinal cord slices.
Studies of central sensitization in the spinal cord have generally
found induction of this phenomenon to be particularly dependent on
repetitive small fiber activation (e.g., Liu and Sandkuhler
1997
). LT is a rostrocaudally extending fiber bundle that
covers the dorsal horn (also called the dorsal root entry zone) and is
known to primarily contain small-diameter primary afferents en route to the dorsal horn where they synapse on intrinsic spinal neurons, particularly those involved in nociception (Chung et al.
1979
). Because electrical stimulation thresholds for large
fibers are lower than those of small fibers, we hypothesized that there
might be differences between evoked potentials from LT and DR stimulation.
Characterization DR and LT stimulation-evoked field potentials
DR and the LT electrodes were stimulated once every minute in
alternating sequence with a 0.5-ms pulse at the current intensity that
produced a half-maximal field potential amplitude (S1/2). In the
presence of bicuculline (10 µM) and strychnine (1 µM), inhibitors
of GABAA and glycine inhibitory
neurotransmission, respectively, both LT and DR stimulation produced
presumably excitatory field potentials whose amplitudes were stable for
many minutes (see Fig. 3A for example and Fig.
2A for group means). Both of these evoked potentials were significantly inhibited by 10 mM MgCl2 (Fig. 2A), suggesting that the
potentials represent a synaptically mediated response. Moreover, the
AMPA and kainate glutamate receptor antagonist CNQX (10 µM) also
inhibited both LT and DR potentials (Fig. 2A), implicating
excitatory amino acids in mediating both of these excitatory responses
[both AMPA (Willcockson et al. 1984a) and
kainate (Li et al. 1999
) glutamate receptors have been
implicated in mediating dorsal horn neurotransmission]. LT- and
DR-evoked potentials did differ from each other, however, in apparent
response latency. At identical stimulus intensities, DR produced a
field potential with an estimated minimum conduction velocity of 3-5 m/s (calculated by the distance between the stimulating electrode and
the recording electrode divided by the latency of the initial field
potential peak; n = 4), whereas the LT-evoked field
potentials had an approximately 0.5-1 m/s minimum conduction velocity
(n = 4), suggesting, as expected, that smaller fibers
may be involved in mediating the LT-evoked responses. The DR- and
LT-evoked field potentials also differed from one another in their
modulation by mu opioids. The LT-, but not the DR-, evoked response was
significantly inhibited by the mu agonist DAMGO (1 µM;
n = 6; Fig. 2B), perhaps indicative of the
greater proportion of LT fibers thought to be involved in nociception
in comparison to the DR (Coggeshall et al. 1981
). The
opioid antagonist naloxone (1 µM) significantly reversed the DAMGO
inhibition of LT-evoked responses without having any effect by itself
(Fig. 2B).
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LT field potentials (e.g., Fig. 3A, inset) were significantly potentiated 30 min after 10-Hz tetanic stimulation (12 1-s trains of 10-Hz 0.5-ms pulses at the S1/2 intensity, given 1 train every 10 s) of LT (Fig. 3, A and B) but not DR (Fig. 3B). Tetanic stimulation of DR (using the same stimulus parameters) produced no significant potentiation of either LT- or DR-evoked potentials (Fig. 3B).
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Pharmacological modulation of LTP of LT-evoked responses
LTP was operationally defined in these studies as the potentiation
of LT-evoked field potentials 30 min following tetanic stimulation.
This time period is longer than those employed in other distinct models
of activity-dependent potentiation including post-tetanic potentiation
and short-term potentiation and has been utilized by ourselves and
others (e.g., Alzheimer et al. 1991; Terman et
al. 1994
) in studies of hippocampal LTP. LTP was also noted at
1 h and 90 min post LTP induction in our initial studies of spinal
LTP. However, since determining the time course of LTP was not a goal
of these experiments, these time points were examined in only a few cells.
Nociceptive sensitization, as mentioned in the preceding text, has been linked to activation of the glutamate NMDA receptor in that NMDA antagonists can prevent sensitization in vivo and in vitro. We studied the role of NMDA receptors in the induction of LT-evoked response LTP. Pretreatment of slices for 10 min with the NMDA antagonist APV (50 µM) had no effect on LT-evoked response amplitudes (data not shown) but significantly inhibited LTP following tetanic LT stimulation (Fig. 4A). This LTP was also inhibited by pretreatment with DAMGO (1 µM; Fig. 4A), an effect antagonized by co-administration of naloxone (1 µM). Naloxone itself, had no effect on LTP (Fig. 4A), suggesting that endogenous opioids have no role in modulating this phenomenon.
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The inhibitory effect of mu opioids on LTP produced by repeated LT stimulation could conceivably be due to effects on LTP induction, expression, or maintenance mechanisms or some combination of these three. To determine which of these mechanisms of LTP are primarily inhibited by DAMGO, we administered DAMGO to three separate groups of slices, followed 10 min later by naloxone. The timing of the DAMGO and naloxone administration differed between groups with respect to the 2-min LTP-inducing stimulus (LT tetanic stimulation). In one group, both DAMGO and naloxone were given 10 min before induction. In another group, DAMGO was given before LTP induction and naloxone was given after it. In the third group of slices, both DAMGO and naloxone were given immediately after the LTP-inducing stimulus.
LTP was inhibited only when DAMGO was bound to opiate receptors (i.e., naloxone was not present) during LTP induction (Fig. 4B), suggesting that it is induction mechanisms with which DAMGO interferes predominantly. The inhibition of induction noted here does not rule out additional mu effects on maintenance and, particularly, expression mechanisms of LTP. Indeed, DAMGO seemed to inhibit to some degree the expression of potentiation prior to naloxone administration when both DAMGO and naloxone were given after LTP induction. Nonetheless data were not routinely collected at this short interval after LTP induction in this or other groups making comparisons impossible. We can conclude, however, that effects on induction are sufficient to explain the inhibition of LTP by mu opioids.
DAMGO effects on LT-evoked EPSCs
The inhibitory effect of DAMGO on LTP induction could be due to
either presynaptic or postsynaptic effects of the opioid agonist. Both
pre- (e.g., Hori et al. 1992; Kangrga and Randic
1991
) and postsynaptic (e.g., Jeftinija 1988
;
Yoshimura and North 1983
) effects of mu opioids on
spinal neurons have been reported previously. Indeed in our studies
using evoked field potentials as a measure of neural activity, both
pre- and postsynaptic effects might occur at the same or different
synapses monitored by our recording electrode. To differentiate such
effects, we utilized the whole cell voltage-clamp technique to record
from specific dorsal horn cells activated synaptically by LT stimulation.
For these studies, cells in Lamina I of the dorsal horn were chosen
because of the known importance of this cell layer in relaying
nociceptive information from primary afferents to supraspinal sites
(Lima et al. 1993) [e.g., nociceptive specific
spinothalamic cells are most common in this spinal cord layer
(Willis and Westlund 1997
)]. As demonstrated above with
LT stimulation-evoked field potentials, LT stimulation-evoked
postsynaptic currents (EPSCs) were also consistently inhibited by DAMGO
(see Fig. 5 for example and Fig.
6B for pooled data) in a
naloxone reversible manner. Again, in the presence of bicuculline and
strychnine, CNQX almost completely eliminated residual postsynaptic
currents (Figs. 5 and 6B) as it had the field potentials. In
these studies, however, the effects of bicuculline and strychnine were
also examined prior to DAMGO application. Bicuculline and strychnine
were both administered to all cells to block
GABAA and glycine-mediated inhibitory
postsynaptic currents (IPSCs) respectively, as before. However, unlike
in previous studies, the two antagonists were added one at a time in a
counterbalanced fashion. All cells were found to have LT activated
bicuculline- or strychnine-sensitive synaptic inputs (the combination
of both drugs inhibited PSC amplitudes by 48 ± 14%, mean ± SD; n = 17; data not shown). A significant
inhibition of PSC amplitudes was produced only when strychnine
(n = 9) was added first to the perfusate (Fig.
6A), although bicuculline also had inhibitory effects in many cells independent of the effects of strychnine on that cell. The
activation of GABA and glycine-mediated IPSCs in Lamina I neurons by LT
stimulation parallels results of others using other primary afferent
stimulation or in other dorsal horn laminae (e.g., Yoshimura and
Nishi 1993
). In the remainder of the studies, bicuculline and
strychnine were routinely added to the perfusate prior to any recording
as in the field potential studies.
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Presynaptic inhibition of LT-evoked EPSCs by DAMGO
In all cells perfused to date with DAMGO under whole cell
voltage-clamp conditions, current/voltage (I-V) curves were
constructed prior to and following DAMGO administration. No naloxone
reversible changes in current amplitudes at any holding potentials
(200-ms, 20-mV steps from 120 to 20 mV) have been observed in any
cell (n = 22; e.g., Fig.
7A).
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The absence of effects of DAMGO on I-V curves in
voltage-clamped Lamina I neurons, suggests that the DAMGO effects are
presynaptic rather than postsynaptic in this region, presumably acting
on mu receptors located on primary afferent terminals to inhibit neurotransmission and perhaps LTP. We also evaluated possible postsynaptic DAMGO inhibitory effects in this region by comparing, in
the same voltage-clamped cell, DAMGO's effects on LT stimulation evoked EPSCs and on excitatory currents evoked by puffing exogenous glutamate near the cell. As before, LT stimulation evoked
CNQX-sensitive EPSC amplitudes were inhibited by DAMGO (by 37 ± 11%, n = 10), in a naloxone-reversible way (e.g., Fig.
7B). However, the amplitude of currents produced by
glutamate puffs were not significantly inhibited by DAMGO (decreased
8 ± 9%, n = 10), although they were significantly attenuated by CNQX (by 72 ± 17%). These data give further evidence for a primarily presynaptic inhibitory effect of DAMGO
on excitatory transmission in Lamina I of the spinal dorsal horn,
probably via inhibition of glutamate release from primary afferent
terminalsproviding a likely mechanism for DAMGO's inhibition of LTP
induction seen in our field potential studies.
The effect of DAMGO on glutamate release was further evaluated using a
paired-pulse stimulation paradigma common technique utilized to
assess the pre- or postsynaptic effects of a neuromodulator (e.g.,
Simmons et al. 1994
). When pairs of stimuli are
delivered in rapid succession, residual calcium in the presynaptic
terminal following the first stimulation leads to an enhanced
transmitter release and a resultant facilitated postsynaptic response
to the second stimulus (e.g., Debanne et al. 1996
).
Paired-pulse facilitation is enhanced when the probability of
neurotransmitter release is relatively low, and thus treatment-induced
alterations in paired-pulse facilitation reflect changes in the
probability of neurotransmitter release from the presynaptic terminal.
Paired-pulse facilitation of LT stimulation-evoked EPSCs was significantly potentiated in Lamina I neurons following DAMGO (1 µM) application (Fig. 8). This effect was reversed by naloxone (1 µM), and both of the paired responses were virtually eliminated by CNQX (10 µM; data not shown) supporting the conclusion that DAMGO acts in this region to decrease the release of glutamate from presynaptic terminals.
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DISCUSSION |
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Much evidence suggests that neurotransmission in the spinal dorsal horn, as in other areas of the CNS, is capable of tremendous activity-dependent plasticity that can cause long-term consequences for subsequent responses to stimuli and thereby sensitize nociceptive responses and perhaps lead to certain chronic pain states. In this manuscript, we report studies of in vitro spinal dorsal horn neurotransmission and LTP following tetanic stimulation of small-diameter primary afferents. We find that LTP but not pretetanus neurotransmission is dependent on NMDA receptors and that both neurotransmission and LTP induction can be inhibited by mu opiate analgesics. In individual dorsal horn cells, mu opioids inhibit glutamate-mediated neurotransmission from primary afferents independent of effects on the postsynaptic cells. This suggests that presynaptic inhibition of glutamate release may be sufficient to explain the inhibitory effects of mu opiates on neurotransmission and LTP induction.
Our findings of reliable and reproducible evoked field potentials in
the rat dorsal horn and LTP of these responses following repeated
primary afferent stimulation have also been reported by others
(Liu and Sandkuhler 1995, 1997
, 1998
; Liu et al.
1997
; Pockett 1995
; Sandkuhler and Liu
1998
). In our studies, electrical stimulation of Lissauer's
tract is particularly effective in producing LTP in comparison to
dorsal root stimulation. Although it is possible that this difference
is due to an LTP inhibitory mechanism (relying neither on glycine nor
GABAA substrates) activated by dorsal root stimulation, our response latency estimates suggest that we are simply
stimulating different sized primary afferents at the two stimulation
sites. Admittedly, the inherent uncertainty in our studies concerning
current spread of the stimulation and the location of neural processes
contributing to the field potentials makes it impossible to compare our
estimates of conduction velocity to those of primary afferents known to
mediate nociception in vivo (C and A
fibers). Moreover decreases in
the degree of myelination in neonatal rat spinal cord compared with the
adult (Chung and Coggeshall 1984
) also make such
comparisons difficult. Nonetheless the differences in field potential
latencies seen here do suggest that LT-evoked field potentials result
from activation of smaller or less myelinated afferent fibers than
DR-evoked potentials and is consistent with the distinct anatomical
compositions of DR and LT (Chung et al. 1979
). Other
investigators have used LT stimulation as a means of activating primary
afferent-superficial dorsal horn synaptic responses (e.g.,
Magnuson and Dickenson 1991
) and the smaller primary
afferents likely stimulated in LT appear particularly important in
mediating nociception (Coggeshall et al. 1981
), inducing sensitization (Baranauskas and Nistri 1998
) and in their
sensitivity to mu opioids [as seen in the present studies and those of
Li et al. (1999)
].
Many in vitro (e.g., King and Thompson 1989; Li
et al. 1999
; Schneider et al. 1998
) and in vivo
(e.g., Dougherty and Willis 1991
; Headley et al.
1987
; Procter et al. 1998
; Radhakrishnan and Henry 1993
; Willcockson et al. 1984a
)
studies of primary afferent-dorsal horn neurotransmission have
identified an important role of glutamate. Indeed, glutamate has been
found to excite nearly 100% of dorsal horn neurons thought to be
involved in pain processing, primarily via AMPA (Dougherty and
Willis 1991
; Headley et al. 1987
;
Willcockson et al. 1984
) and, more recently, kainate
(Li et al. 1999
; Procter et al. 1998
)
receptors. Thus our finding that the AMPA antagonist CNQX greatly
diminished both LT and DR stimulation evoked responses is not
surprising. Recently ATP acting at P2X receptors on spinal neurons
(dissociated or in culture) has also been implicated in "fast
transmission" (Bardoni et al. 1997
; Gu and
MacDermott 1997
) perhaps relevant to nociception
(Bland-Ward and Humphrey 1997
; Cook et al.
1997
) [although this is controversial (Li et al.
1998
)]. Whether the very small CNQX-resistant components of
evoked potentials and Lamina I EPSCs observed in our studies were
mediated by ATP was not specifically examined. However, in the field
potential studies, the CNQX-resistant responses were not significantly
different from those observed in high magnesium buffer, suggesting that this technique has little sensitivity for studying nonglutamatergic fast synaptic transmission. Such fast transmission is in contrast to
slower onset NMDA receptor-mediated glutamate actions within the dorsal
horn (King and Lopez-Garcia 1993
), which normally follow initial activation of AMPA receptors (Jeftinija and Urban
1994
; Randic et al. 1993
; Svendsen et al.
1998
) and are consistent with a role for NMDA receptors in
mediating longer latency plastic changes (as seen in our LTP studies
here). Mu opiates have been reported capable of inhibiting both AMPA
(Hori et al. 1992
) and NMDA (Jeftinija
1988
; Willcockson et al. 1984b
)
receptor-mediated primary afferent neurotransmission in the dorsal horn
via both pre- (Hori et al. 1992
) and postsynaptic
(Jeftinija 1988
) mechanisms.
The inhibition of LTP induction by DAMGO and, particularly, its
inhibition of effects from synaptically released glutamate (but not
exogenous glutamate) suggests that DAMGO acts by inhibiting presynaptic
glutamate release. This suggestion is strengthened by our observation
of a naloxone-sensitive DAMGO-induced enhancement of paired-pulse
facilitation. Others have demonstrated presynaptic opioid effects on
excitatory dorsal horn synaptic transmission (Glaum et al.
1994; Hori et al. 1992
; Jeftinija
1988
; Kohno et al. 1999
). For example,
Kohno et al. (1999)
reported inhibitory effects of DAMGO
on glutamatergic EPSCs in Lamina II (substantia gelatinosa) cells. In
addition, they found that miniature EPSC frequency was also inhibited,
further supporting the hypothesis that these effects were due to a
decrease in glutamate release. The molecular mechanisms underlying
these effects are not known. Both an inhibition of calcium channels and
an activation of potassium channels have been suggested to mediate mu
opiate receptor effects in the spinal cord (Duggan and North
1983
; Schneider et al. 1998
; Schroeder et
al. 1991
; Taddese et al. 1995
). Although we were unable to document any postsynaptic effects of DAMGO, such effects have
also been reported in the rat superficial dorsal horn (Yoshimura and North 1983
). It is important to note however, that these
effects have been most commonly reported in Lamina II cells and not the Lamina I cells studied here. Indeed there appear to be no reports of
Lamina I postsynaptic effects, which is again consistent with the
related anatomical literature. Although rhizotomy studies have found
that half of dorsal horn mu opiate receptors are intrinsic to the
spinal cord (Coggeshall and Carlton 1997
),
Cheng et al., using ultrastructural analysis, have localized just 12%
of superficial dorsal horn mu receptors to somata or dendrites
(Cheng et al. 1997
). This suggests that the remaining mu
receptors are located on axon terminals of spinal interneurons and
concurs with the low incidence of postsynaptic neurophysiological
effects seen in the literature, in general, and in our own work, in particular.
The presynaptic effects of mu opiates in inhibiting LTP induction in
the spinal cord parallel our findings in the dentate region of the
hippocampus that kappa opiates inhibit LTP induction (Terman et
al. 1994; Wagner et al. 1993
). Moreover we have
found in the dentate that the endogenous kappa opioids, dynorphins, are
released from postsynaptic neurons during repeated afferent stimulation
and act presynaptically to modulate LTP (Simmons et al.
1994
). No evidence of endogenous opioid effects were unmasked in the present studies by naloxone, although Pockett has previously reported (Pockett 1995
) naloxone-induced inhibition of
long-term depression of field potentials in the spinal cord. Indeed
preliminary evidence from our laboratory suggests that much like the
hippocampus, dynorphins in the spinal cord may provide feedback
inhibition to modulate LTP. The kappa1 opiate
receptor antagonist norbinaltorphimine, significantly potentiates LTP
of Lamina I EPSCs (Taylor et al. 1998
). In addition,
other mechanisms appear to provide feedback inhibition for LTP in the
spinal cord. In a series of studies, Sandkuhler's group has
investigated in vivo the underlying pharmacology and necessary stimulus
parameters for inducing dorsal horn field potential LTP evoked by
primary afferent stimulation (Liu and Sandkuhler 1995
, 1997
,
1998
; Liu et al. 1998
; Sandkuhler and Liu 1998
). Although C-fiber stimulation and resultant release of
neurokinins have been found to be critical for inducing LTP in their
studies, both A-delta primary afferent activity (Liu et al.
1998
) and descending inhibitory mechanisms (Sandkuhler
and Liu 1998
) can powerfully inhibit such LTP.
The importance of spinal LTP in mediating central sensitization
reported in laboratory animals and man is not known. A number of other
mechanisms have been proposed to account for changes in sensitivity of
spinal nociceptive neurons to stimulation. These include decreases in
intrinsic (Wiesenfeld-Hallin et al. 1997) or descending
inhibition (Sandkuhler and Liu 1998
), increases in
descending facilitation (and facilitatory) (Urban et al.
1999
) and increases in numbers of excitatory synaptic
connections (e.g., Woolf and Doubell 1994
; Woolf
et al. 1992
, 1995
). Nonetheless, pretreatment with mu opioids
have been found effective in blocking exacerbations of postoperative
(Katz et al. 1992
) and chronic pain (Bach et al.
1988
) and has led to the concept of "preemptive analgesia"
(Katz et al. 1992
) as a strategy to avoid
activity-dependent neuroplastic changes in nociception. The current
studies of primary afferent LTP in the spinal cord support the idea of
aggressive treatment of nociception with mu opiates in an effort to
inhibit LTP induction.
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ACKNOWLEDGMENTS |
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We thank Dr. Albert Berger of the Department of Physiology and Biophysics at the University of Washington for his help during the course of these studies.
This work was supported by National Institute on Drug Abuse Grants DA-00266 (G. W. Terman) and DA-04123 (C. Chavkin).
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FOOTNOTES |
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Address for reprint requests: G. Terman, Dept. of Anesthesiology, Box 356540, University of Washington, Seattle, WA 98195-6540 (E-mail: gwt{at}u.washington.edu).
Received 17 December 1999; accepted in final form 18 October 2000.
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REFERENCES |
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